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Validation of reference genes for qPCR studies on Caco-2 cell differentiation
Validation of reference gene expression stabilities is a prerequisite for reliable normalization of qPCR data. The present study assessed the variation of six reference genes (ACTB, GAPDH, B2M, HPRT1, SDHA, YWHAZ) in Caco-2 cells under the influence of different growth supports and cultivation periods. Genes were ranked according to their stability using the geNorm software. To verify the influence of
Claudia Piana; Michael Wirth; Stefan Gerbes; Helmut Viernstein; Franz Gabor; Stefan Toegel
Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks
BACKGROUND: Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined
Kalle T Rytkönen; Gillian MC Renshaw; Kevin J Ashton; Grant Williams-Pritchard; Erica H Leder; Mikko Nikinmaa
Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR
BACKGROUND: Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. RESULTS: In
Ann-Britt Nygard; Claus B Jřrgensen; Susanna Cirera; Merete Fredholm
Reference gene validation for qPCR in rat carotid body during postnatal development
Background The carotid bodies are the main arterial oxygen chemoreceptors in mammals. Afferent neural output from the carotid bodies to brainstem respiratory and cardiovascular nuclei provides tonic input and mediates important protective responses to acute and chronic hypoxia. It is widely accepted that the selection of reference genes for mRNA normalization in quantitative real-time PCR must be validated for a given tissue and set of conditions. This is particularly important for studies in carotid body during early postnatal maturation as the arterial oxygen tension undergoes major changes from fetal to postnatal life, which may affect reference gene expression. In order to determine the most stable and suitable reference genes for the study of rat carotid body during development, six commonly used reference genes, ?-actin, RPII (RNA polymerase II), PPIA (peptidyl-proyl-isomerase A), TBP (TATA-box binding protein), GAPDH, and 18s rRNA, were evaluated in two age groups (P0-1 and P14-16) under three environmental oxygen conditions (normoxia, chronic hypoxia and chronic hyperoxia) using the three most commonly used software programs, geNorm, NormFinder and BestKeeper. Findings The three programs produced similar results but the reference gene rankings were not identical between programs or experimental conditions. Overall, 18s rRNA was the least stable reference gene for carotid body and, when hyperoxia and/or hypoxia conditions were included, actin was similarly unstable. Conclusions Reference or housekeeping gene expression for qPCR studies of carotid body during postnatal development may vary with developmental stage and environmental conditions. Selection of the best reference gene or combination of reference genes for carotid body development studies should take environmental conditions into account. Two commonly used reference genes, 18s rRNA and actin, may be unsuitable for studies of carotid body maturation, especially if the study design includes altered oxygen conditions.
Selection of reference genes for qPCR in hairy root cultures of peanut
Background Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. Results A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). Conclusions This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.
Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis
The selection and validation of reference genes constitute a key point for gene expression analysis based on qPCR, requiring\\u000a efficient normalization approaches. In this work, the expression profiles of eight genes were evaluated to identify novel\\u000a reference genes for transcriptional studies associated to the senescence process in sunflower. Three alternative strategies\\u000a were applied for the evaluation of gene expression stability
Paula FernandezJulio; Julio A. Di Rienzo; Sebastián Moschen; Guillermo A. A. Dosio; Luis A. N. Aguirrezábal; H. Esteban Hopp; Norma Paniego; Ruth A. Heinz
Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree
Real-time reverse transcriptase PCR is a powerful tool to investigate relevant changes in gene expression during plant somatic embryogenesis (S.E.); however, this method lacks ideal reference genes. To select the most stable reference genes for S.E. studies, the expression profiles of seven frequently used reference genes (18S RNA, eIF-4a, UBQ, ACTB, EF-1a, Histone H3, and 2-TUB) and functional genes (Fe-SOD,
Y. L. Lin; Z. X. Lai
Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes
The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions.
Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt
Selection of reliable reference genes for qPCR studies on chondroprotective action
BACKGROUND: Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected
Stefan Toegel; Wenwen Huang; Claudia Piana; Frank M Unger; Michael Wirth; Mary B Goldring; Franz Gabor; Helmut Viernstein
Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass ( Dicentrarchus labrax)
The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to
Karin Mitter; Georgios Kotoulas; Antonios Magoulas; Victor Mulero; Pilar Sepulcre; Antonio Figueras; Beatrice Novoa; Elena Sarropoulou
Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: is ?-actin a reliable normalizer for photoaging studies?
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or ?-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/?2M were the most stable ones under hypoxia in HDF exposed to 18 J/cm(2) UVA. ?-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF. PMID:21963587
Brugč, F; Venditti, E; Tiano, L; Littarru, G P; Damiani, E
Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation.
The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ??Cq/Normfinder and ??Cq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2 Gy, and changes in gene expression were analyzed 24 h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ??Cq/Genorm or ??Cq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested. PMID:23884658
Reuther, Sebastian; Reiter, Martina; Raabe, Annette; Dikomey, Ekkehard
Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue
Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous\\u000a reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference\\u000a genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase,\\u000a TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate\\u000a dehydrogenase complex, subunit
Antje Koppelkamm; Benedikt Vennemann; Tony Fracasso; Sabine Lutz-Bonengel; Ulrike Schmidt; Marielle Heinrich
Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is ?-actin a reliable normalizer for photoaging studies?
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or ?-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both
F. Brugč; E. Venditti; L. Tiano; G. P. Littarru; E. Damiani
qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL
Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST.
Selecting control genes for RT-QPCR using public microarray data
Background Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. Results We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at Conclusion We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable.
Popovici, Vlad; Goldstein, Darlene R; Antonov, Janine; Jaggi, Rolf; Delorenzi, Mauro; Wirapati, Pratyaksha
Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells
The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR.
Livak, Kenneth J.; Wills, Quin F.; Tipping, Alex J.; Datta, Krishnalekha; Mittal, Rowena; Goldson, Andrew J.; Sexton, Darren W.; Holmes, Chris C.
Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells.
The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR. PMID:23079396
Livak, Kenneth J; Wills, Quin F; Tipping, Alex J; Datta, Krishnalekha; Mittal, Rowena; Goldson, Andrew J; Sexton, Darren W; Holmes, Chris C
Selecting control genes for RT-QPCR using public microarray data
BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different
Vlad Popovici; Darlene R. Goldstein; Janine Antonov; Rolf Jaggi; Mauro Delorenzi; Pratyaksha Wirapati
Measurement of copepod predation on nauplii using qPCR of the cytochrome oxidase I gene
A method to directly measure predation rates by older stage copepods upon copepod nauplii using species-specific primers for\\u000a the mitochondrial cytochrome oxidase subunit one gene (mtCOI) and real-time quantitative PCR (qPCR) was developed. The general\\u000a approach is to determine the mtCOI gene copy number of an individual prey organism and the copy number of the same gene in\\u000a the stomachs
Edward G. Durbin; Maria C. Casas; Tatiana A. Rynearson; David C. Smith
Evaluation of Quantitative PCR Reference Genes for Gene Expression Studies in Tribolium castaneum After Fungal Challenge
Technology Transfer Automated Retrieval System (TEKTRAN)
To investigate gene expression in Tribolium castaneum exposed to Beauveria bassiana, reference genes for qPCR were evaluated. Of these, the widely used genes for ß-actin, a-tubulin, and RPS6 were not stable. The most stable were ribosomal protein genes, RPS3, RPS18, and RPL13a. Syntaxin1, syntaxin6...
Comprehensive qPCR profiling of gene expression in single neuronal cells.
A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe uses Fluidigm Biomark dynamic arrays for high-throughput expression profiling from single neuronal cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment, which can be completed within 2-3 d. The protocol enables simple and cost-effective profiling of several hundred transcripts from a single cell, and it could have numerous utilities. PMID:22193304
Citri, Ami; Pang, Zhiping P; Südhof, Thomas C; Wernig, Marius; Malenka, Robert C
Selection of reference genes for quantitative real-time RT-PCR analysis in citrus
Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying\\u000a low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an\\u000a internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression\\u000a levels. Expression of the reference gene should be
Jiawen Yan; Feirong Yuan; Guiyou Long; Lei Qin; Ziniu Deng
Design and Application of an Internal Amplification Control to Improve Dehalococcoides mccartyi 16S rRNA Gene Enumeration by qPCR.
Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of chlorinated ethenes to nontoxic ethene in contaminated aquifers. Enumeration of Dhc biomarker genes using quantitative real-time PCR (qPCR) in groundwater is a key component of site assessment and bioremediation monitoring. Unfortunately, standardized qPCR procedures that recognize impaired measurements due to PCR inhibition, low template DNA concentrations, or analytical error are not available, thus limiting confidence in qPCR data. To improve contemporary approaches for enumerating Dhc in environmental samples, multiplex qPCR assays were designed to quantify the Dhc 16S rRNA gene and one of two different internal amplification controls (IACs): a modified Dhc 16S rRNA gene fragment (Dhc*) and the firefly luciferase gene luc. The Dhc* IAC exhibited competitive inhibition in qPCR with the Dhc 16S rRNA gene template when the ratio of either target was 100-fold greater than the other target. A multiplex qPCR assay with the luc IAC avoided competitive inhibition and accurately quantified Dhc abundances ranging from ?10 to 10(7) 16S rRNA gene copies per reaction. The addition of ?10(6) E. coli luc IAC to simulated groundwater amended with the Dhc-containing consortium KB-1 yielded reproducible luc counts after DNA extraction and multiplex qPCR enumeration. The application of the luc IAC assay improved Dhc biomarker gene quantification from simulated groundwater samples and is a valuable approach for "ground truthing" qPCR data obtained in different laboratories, thus reducing ambiguity associated with qPCR enumeration and reproducibility. PMID:24053159
Hatt, Janet K; Ritalahti, Kirsti M; Ogles, Dora M; Lebrón, Carmen A; Löffler, Frank E
Evaluation of potential reference genes for use in gene expression studies in the conifer pathogen (Heterobasidion annosum).
The basidiomycete Heterobasidion annosum is the causative agent of butt and root rot disease of conifer trees and it's one of the most destructive conifer pathogen in the northern hemisphere. Because of the intrinsic difficulties in genome manipulation in this fungus, most studies have been focused on gene expression analysis using quantitative real time polymerase chain reaction (qPCR). qPCR is a powerful technique but its reliability resides in the correct selection of a set of reference genes used in the data normalization. In this study, we determined the expression stability of 11 selected reference genes in H. annosum. Almost nothing has so far been published about validation of a set of reference genes to be used in gene expression experiments in this fungus. Eleven reference genes were validated in H. annosum which was grown on three different substrates: pine bark, pine heartwood, and pine sapwood. Bestkeeper and NormFinder Excel-based software were used to evaluate the reference gene transcripts' stability. The results from these two programs indicated that three reference genes namely Tryp metab, RNA Pol3 TF, and Actin were stable in H. annosum in the conditions studied. Interestingly, the GAPDH transcript which has been extensively used in qPCR data normalization is not the best choice when a wide reference gene selection is available. This work represents the first extensive validation of reference genes in H. annosum providing support for gene expression studies and benefits for the wider forest pathology community. PMID:23645035
Raffaello, Tommaso; Asiegbu, Fred O
Superior cross-species reference genes: a blueberry case study.
The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the application of high throughput gene expression approaches that should relatively quickly increase our understanding of blueberry physiology. These studies, however, require a highly accurate and robust workflow and make necessary the identification of reference genes with high expression stability for correct target gene normalization. To create a set of superior reference genes for blueberry expression analyses, we mined a publicly available transcriptome data set from blueberry for orthologs to a set of Arabidopsis genes that showed the most stable expression in a developmental series. In total, the expression stability of 13 putative reference genes was evaluated by qPCR and a set of new references with high stability values across a developmental series in fruits and floral buds of blueberry were identified. We also demonstrated the need to use at least two, preferably three, reference genes to avoid inconsistencies in results, even when superior reference genes are used. The new references identified here provide a valuable resource for accurate normalization of gene expression in Vaccinium spp. and may be useful for other members of the Ericaceae family as well. PMID:24058469
Die, Jose V; Rowland, Lisa J
Superior Cross-Species Reference Genes: A Blueberry Case Study
The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the application of high throughput gene expression approaches that should relatively quickly increase our understanding of blueberry physiology. These studies, however, require a highly accurate and robust workflow and make necessary the identification of reference genes with high expression stability for correct target gene normalization. To create a set of superior reference genes for blueberry expression analyses, we mined a publicly available transcriptome data set from blueberry for orthologs to a set of Arabidopsis genes that showed the most stable expression in a developmental series. In total, the expression stability of 13 putative reference genes was evaluated by qPCR and a set of new references with high stability values across a developmental series in fruits and floral buds of blueberry were identified. We also demonstrated the need to use at least two, preferably three, reference genes to avoid inconsistencies in results, even when superior reference genes are used. The new references identified here provide a valuable resource for accurate normalization of gene expression in Vaccinium spp. and may be useful for other members of the Ericaceae family as well.
Die, Jose V.; Rowland, Lisa J.
Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut
The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.
Cindhuri, Katamreddy Sri; Sharma, Kiran K.
Identification of condition-specific reference genes from microarray data for locusts exposed to hypobaric hypoxia.
Real-time quantitative polymerase chain reaction (qPCR) is a routine and robust approach for measuring gene expression. The stability of reference genes in qPCR is crucial for the accurate quantification of gene expression. To provide reliable reference genes for studying the transcriptional responses of locust muscles to hypobaric hypoxia, we first examined the gene expression stability of the frequently used housekeeping genes 18S, GAPDH, and ?-actin. However, the expression of these three housekeeping genes was influenced by hypobaric hypoxia. Consequently, we identified five novel candidate reference genes from the locust microarray data. The gene expression stability of the five candidates, together with the three classical housekeeping genes, were evaluated using two distinct algorithms implemented in geNorm and NormFinder. GeNorm identified Ach (acetyl-CoA hydrolase) and Pgp (phosphoglycolate phosphatase-like) as the most stable genes and NormFinder further distinguished Ach as the most stable one. The validity of Ach as a reference gene was confirmed through comparison with 18S. This study exemplifies the necessity of validating reference genes before their application and the feasibility of identifying condition-specific reference genes from large-scale gene expression data. PMID:23650605
Zhao, De-Jian; Guo, Kun; Kang, Le
Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).
We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ?-actin (ACTB), ?-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ?-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation. PMID:22612931
Varshney, N; Mohanty, A K; Kumar, S; Kaushik, J K; Dang, A K; Mukesh, M; Mishra, B P; Kataria, R; Kimothi, S P; Mukhopadhyay, T K; Malakar, D; Prakash, B S; Grover, S; Batish, V K
Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol
Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psm?1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.
Franca, Angela; Freitas, Ana I.; Henriques, Ana F.; Cerca, Nuno
A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional ?-Thalassemia.
The predominant determinants of ?-thalassemia are deletions in the human ?-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human ?-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six ?-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-??Cq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for ?-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening. PMID:23810501
Zhou, Wanjun; Wang, Ge; Zhao, Xuefeng; Xiong, Fu; Zhou, Shaoxiong; Peng, Jianming; Cheng, Youming; Xu, Shun; Xu, Xiangmin
Comparison of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Analysis of Gene Expression in Sugarcane
A protocol for reverse transcription followed by real-time quantitative PCR (RT- qPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for ?-actin, ?-tubulin, and
Comprehensive qPCR profiling of gene expression in single neuronal cells
A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe uses Fluidigm Biomark
Thomas C Südhof; Marius Wernig; Ami Citri; Zhiping P Pang; Robert C Malenka
Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition
In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target CsNRT1.1 gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH3). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EF? proved to be the most suitable for normalization of CsNRT1.1 expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.
Warzybok, Anna; Migocka, Magdalena
Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition.
In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target CsNRT1.1 gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH3). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EF? proved to be the most suitable for normalization of CsNRT1.1 expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress. PMID:24058446
Warzybok, Anna; Migocka, Magdalena
Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.
Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1?, FBOX, GAPDH, GTPB, PP2A, SAND, TUB?, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. PMID:23954326
Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang
Determination of reference genes for circadian studies in different tissues and mouse strains
Background Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration. Results In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons. Conclusions Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.
Validation of a set of reference genes to study response to herbicide stress in grasses
Background Non-target-site based resistance to herbicides is a major threat to the chemical control of agronomically noxious weeds. This adaptive trait is endowed by differences in the expression of a number of genes in plants that are resistant or sensitive to herbicides. Quantification of the expression of such genes requires normalising qPCR data using reference genes with stable expression in the system studied as internal standards. The aim of this study was to validate reference genes in Alopecurus myosuroides, a grass (Poaceae) weed of economic and agronomic importance with no genomic resources. Results The stability of 11 candidate reference genes was assessed in plants resistant or sensitive to herbicides subjected or not to herbicide stress using the complementary statistical methods implemented by NormFinder, BestKeeper and geNorm. Ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase were identified as the best reference genes. The reference gene set accuracy was confirmed by analysing the expression of the gene encoding acetyl-coenzyme A carboxylase, a major herbicide target enzyme, and of an herbicide-induced gene encoding a glutathione-S-transferase. Conclusions This is the first study describing a set of reference genes (ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase) with a stable expression under herbicide stress in grasses. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in grasses.
Identification of four soybean reference genes for gene expression normalization
Technology Transfer Automated Retrieval System (TEKTRAN)
Gene expression analysis requires the use of reference genes stably expressed independently of specific tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin and elongation factor 1-alpha) are commonly used as reference genes with the assumption tha...
Reference Gene Identification for Reverse Transcription-Quantitative Polymerase Chain Reaction Analysis in an Ischemic Wound-Healing Model
Reference genes are often used in RT-quantitative PCR (qPCR) analysis to normalize gene expression levels to a gene that is expressed stably across study groups. They ultimately serve as a control in RT-qPCR analysis, producing more accurate interpretation of results. Whereas many reference genes have been used in various wound-healing studies, the most stable reference gene for ischemic wound-healing analysis has yet to be identified. The goal of this study was to determine systematically the most stable reference gene for studying gene expression in a rat ischemic wound-healing model using RT-qPCR. Twelve commonly used reference genes were analyzed using RT-qPCR and geNorm data analysis to determine stability across normal and ischemic skin tissue. It was ultimately determined that Ubiquitin C (UBC) and ?-2 Microglobulin (B2M) are the most stably conserved reference genes across normal and ischemic skin tissue. UBC and B2M represent reliable reference genes for RT-qPCR studies in the rat ischemic wound model and are unaffected by sustained tissue ischemia. The geometric mean of these two stable genes provides an accurate normalization factor. These results provide insight on dependence of reference-gene stability on experimental parameters and the importance of such reference-gene investigations.
Ruedrich, Elizabeth D.; Henzel, Mary K.; Hausman, Bryan S.
Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR
Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization\\u000a with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with\\u000a limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in\\u000a genetics
Fernanda Cruz; Samara Kalaoun; Paula Nobile; Carlos Colombo; Juliana Almeida; Leila M. G. Barros; Eduardo Romano; Maria Fátima Grossi-de-Sá; Maité Vaslin; Marcio Alves-Ferreira
Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects
Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ?Ct analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2-??Ct method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.
Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei
Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.
Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ?Ct analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2(-??Ct) method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR. PMID:22938136
Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei
Validation of reference genes for real-time PCR of reproductive system in the black tiger shrimp.
Gene expression of reproductive system of the black tiger shrimp (Peneaus monodon) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for P. monodon reproductive organs is lacking. In this study, the stability of four potential reference genes (18s rRNA, GAPDH, ?-actin, and EF1-?) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-? and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-? were for ovaries from wild-caught broodstock and domesticated groups. EF1-? and ?-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-? and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-? and ?-actin were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, Dmc1 and Vitellogenin, were used to validate these reference genes. When normalized to EF1-?, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-? is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon. PMID:23285145
Leelatanawit, Rungnapa; Klanchui, Amornpan; Uawisetwathana, Umaporn; Karoonuthaisiri, Nitsara
Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
Background Quantitative real-time PCR (qPCR) is a commonly used technique to quantify gene expression levels. Validated normalization is essential to obtain reliable qPCR data. In that context, normalizing to multiple reference genes has become the most popular method. However, expression of reference genes may vary per tissue type, developmental stage and in response to experimental treatment. It is therefore imperative to determine stable reference genes for a specific sample set and experimental model. The present study was designed to validate potential reference genes in hippocampal tissue from rats that had experienced early-life febrile seizures (FS). To this end, we applied an established model in which FS were evoked by exposing 10-day old rat pups to heated air. One week later, we determined the expression stability of seven frequently used reference genes in the hippocampal dentate gyrus. Results Gene expression stability of 18S rRNA, ActB, GusB, Arbp, Tbp, CycA and Rpl13A was tested using geNorm and Normfinder software. The ranking order of reference genes proposed by geNorm was not identical to that suggested by Normfinder. However, both algorithms indicated CycA, Rpl13A and Tbp as the most stable genes, whereas 18S rRNA and ActB were found to be the least stably expressed genes. Conclusions Our data demonstrate that the geometric averaging of at least CycA, Rpl13A and Tbp allows reliable interpretation of gene expression data in this experimental set-up. The results also show that ActB and 18S rRNA are not suited as reference genes in this model.
Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR
Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides
Richard A. Haugland; Manju Varma; Mano Sivaganesan; Catherine Kelty; Lindsay Peed; Orin C. Shanks
Hepatic reference gene selection in adult and juvenile female Atlantic salmon at normal and elevated temperatures
Background The use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity and apparent ease of use. However, experimental error can be introduced at many stages during sample processing and analysis, and for this reason qPCR data are often normalised to an internal reference gene. The present study used three freely available algorithms (GeNorm, NormFinder and BestKeeper) to assess the stability of hepatically expressed candidate reference genes (Hprt1, Tbp, Ef1? and ?-tubulin) in two experiments. In the first, female Atlantic salmon (Salmo salar) broodstock of different ages were reared at either 14 or 22°C for an entire reproductive season, therefore a reference gene that does not respond to thermal challenge or reproductive condition was sought. In the second, estrogen treated juvenile salmon were maintained at the same temperatures for 14 days and a reference gene that does not respond to temperature or estrogen was required. Additionally, we performed independent statistic analysis to validate the outputs obtained from the program based analysis. Results Based on the independent statistical analysis performed the stability of the genes tested was Tbp > Ef1? > Hprt1 > ?-tubulin for the temperature/reproductive development experiment and Ef1? > Hprt1 > Tbp for the estrogen administration experiment (?-tubulin was not analysed). Results from the algorithms tested were quite ambiguous for both experiments; however all programs consistently identified the least stable candidate gene. BestKeeper provided rankings that were consistent with the independent analysis for both experiments. When an inappropriate candidate reference gene was used to normalise the expression of a hepatically expressed target gene, the ability to detect treatment-dependent changes in target gene expression was lost for multiple groups in both experiments. Conclusions We have highlighted the need to independently validate the results of reference gene selection programs. In addition, we have provided a reference point for those wishing to study the effects of thermal challenge and/or hormonal treatment on gene stability in Atlantic salmon and other teleost species.
Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data
Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1?5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, Gh?TUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; GhACT4 and GhUBQ14 for flower development, GhACT4 and GhFBX6 for the floral organs and GhMZA and GhPTB for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.
Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars.
The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1?, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1? and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1?, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1?, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola. PMID:24023800
Monteiro, Filipa; Sebastiana, Mónica; Pais, Maria Salomé; Figueiredo, Andreia
Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1?, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1? and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1?, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1?, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola.
Monteiro, Filipa; Sebastiana, Monica; Pais, Maria Salome; Figueiredo, Andreia
Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity
Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.
Lardizabal, Maria N.; Nocito, Ana L.; Daniele, Stella M.; Ornella, Leonardo A.; Palatnik, Javier F.; Veggi, Luis M.
Genetics Home Reference: What is gene therapy?
... How Genes Work Mutations and Health Inheritance Consultation Testing Therapy Human Genome Project Genomic Research Next Handbook > Gene Therapy > What is gene therapy? Gene therapy is an ... are testing several approaches to gene therapy, including: Replacing a ...
Blockade of ?2-adrenoceptors induces Arc gene expression in rat brain in a glutamate receptor-dependent manner: a combined qPCR, in situ hybridisation and immunocytochemistry study.
Studies of 5-HT-glutamate interactions suggest that activation of brain 5-HT(2A) receptors leads to an AMPA receptor-mediated induction of the immediate early (activity-dependent) gene, Arc (Arg3.1). In this respect, noradrenaline-glutamate interactions are poorly characterised. Here we investigated the influence on regional brain Arc gene expression of selective blockade of ?(2)-adrenoceptors in rats. Several complementary techniques were used: qPCR (mRNA, discrete tissue punches), in situ hybridisation (mRNA, sections) and immunocytochemistry. The ?(2)-adrenoceptor antagonist, RX 821002, dose-dependently and time-dependently (maximal effect 2 h) increased Arc mRNA levels as demonstrated both by qPCR and in situ hybridisation. The ?(2)-adrenoceptor antagonist, atipamezole, also increased Arc mRNA in in situ hybridisation studies. Changes in Arc mRNA after RX 821002 were of similar magnitude in punches and intact tissue sections and region-specific, with effects being most pronounced in parietal cortex and caudate putamen, less robust in frontal cortex, and not detectable in hippocampal sub-regions. Both qPCR and in situ hybridisation studies demonstrated that RX 821002-induced Arc mRNA was blocked by the AMPA antagonist, GYKI 52466. Pretreatment with the NMDA antagonist MK 801 also prevented RX 821002-induced Arc mRNA, as did the mGluR5 antagonist MPEP, whilst the mGluR2/3 antagonist, LY341495, had no effect. Finally, immunocytochemical studies showed that RX 821002 increased Arc-immunoreactivity in cells in close apposition to ?(2)-adrenoceptor-positive processes. Thus, employing three complementary techniques, these observations demonstrate that blockade of ?(2)-adrenoceptors triggers brain expression of the immediate early gene, Arc, and that this effect involves the recruitment of AMPA, NMDA and mGluR5 but not mGluR2/3 glutamatergic receptors. PMID:22828637
Serres, Florence; Rodriguez, Marianne; Rivet, Jean-Michel; Galizzi, Jean-Pierre; Lockhart, Brian; Sharp, Trevor; Millan, Mark J
Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis.
The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic pterostilbene, which plays a crucial role in grapevine interaction with Plasmopara viticola and Botrytis cinerea. As a resveratrol O-methyltransferase (ROMT) gene involved in pterostilbene synthesis was recently identified, we investigated the accumulation of the corresponding transcripts to those of two other stilbene biosynthesis related genes phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) in response to pathogen infection. Using three computer-based statistical methods and C(t) values or LRE method generated values as input data, we have first identified two reference genes (VATP16 and 60SRP) suitable for normalization of qPCR expression data obtained in grapevine leaves and berries infected by P. viticola and B. cinerea, respectively. Next, we have highlighted that the expression of ROMT is induced in P. viticola-infected leaves and also in B. cinerea-infected berries, confirming the involvement of pterostilbene in grapevine defenses. PMID:21340517
Gamm, Magdalena; Héloir, Marie-Claire; Kelloniemi, Jani; Poinssot, Benoît; Wendehenne, David; Adrian, Marielle
Selection of reliable reference genes for gene expression studies in peach using real-time PCR
BACKGROUND: RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for
Zhaoguo Tong; Zhihong Gao; Fei Wang; Jun Zhou; Zhen Zhang
Monitoring dark hydrogen fermentation performance of indigenous Clostridium butyricum by hydrogenase gene expression using RT-PCR and qPCR
Hydrogenase is the key enzyme responsible for H2 production in dark fermentation. Therefore, the expression of hydrogenase gene may be a good indicator for the performance of a dark H2 fermentation culture. In this study, we investigated the correlation between expression of the functional gene (hydA encoding for hydrogenase in Clostridium butyricum) and bioH2 production activity during batch growth of
Mei-Yun Wang; Yu-Li Tsai; Betty H. Olson; Jo-Shu Chang
Impact of reference gene selection for type 2 cannabinoid receptor gene expression studies in human spermatozoa.
Quantitative real-time polymerase chain reaction (qRT-PCR) has been employed to study the gene expression profiles in human spermatozoa, but accurate analysis is dependent upon normalisation of data against an endogenous control. ?-Actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are the most commonly used reference genes for normalisation of gene expression in human spermatozoa, but the expression of these genes in many tissues has considerable variation under different physiological, pathological and experimental conditions which limits their effectiveness in normalisation. The expression stability of a panel of 12 reference genes was studied in normal and pathological human spermatozoa using geNorm and NormFinder software. Although there were some discrepancies in the ranking of reference gene stability, each software program ranked B2 M, ACTB, CYC1 and 18S RNA within the top 5 and recommended the combined use of at least two reference genes. Normalisation of qRT-PCR data for the cannabinoid receptor type 2 in spermatozoa using the different housekeeping genes demonstrated how, without validation, conflicting results are obtained. We recommend that the arbitrary use of reference genes should be avoided and the validation of reference gene stability should be undertaken prior to every study. For normalisation of CB2 expression, we would recommend using the geometric mean of B2 M and ACTB. PMID:22928818
Amoako, A A; Gebeh, A K; Marczylo, E L; Willets, J M; Elson, J; Marczylo, T H; Konje, J C
Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine
Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1\\/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of
Nuria Hierro; Braulio Esteve-Zarzoso; Angel Gonzalez; Albert Mas; Jose M. Guillamon
Expression stability of commonly used reference genes in canine articular connective tissues
BACKGROUND: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Real-time reverse transcription PCR and a
Duncan Ayers; Dylan N Clements; Fiona Salway; Philip JR Day
AutDB: a gene reference resource for autism research
Recent advances in studies of Autism Spectrum Disorders (ASD) has uncovered many new candidate genes and continues to do so at an accelerated pace. To address the genetic complexity of ASD, we have developed AutDB (http://www.mindspec.org/autdb.html), a publicly available web-portal for on-going collection, manual annotation and visualization of genes linked to the disorder. We present a disease-driven database model in AutDB where all genes connected to ASD are collected and classified according to their genetic variation: candidates identified from genetic association studies, rare single gene mutations and genes linked to syndromic autism. Gene entries are richly annotated for their relevance to autism, along with an in-depth view of their molecular functions. The content of AutDB originates entirely from the published scientific literature and is organized to optimize its use by the research community. The main focus of this resource is to provide an up-to-date, annotated list of ASD candidate genes in the form of reference dataset for interrogating molecular mechanisms underlying the disorder. Our model for consolidated knowledge representation in genetically complex disorders could be replicated to study other such disorders.
Basu, Saumyendra N.; Kollu, Ravi; Banerjee-Basu, Sharmila
Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution.
This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. PMID:20079846
Weaver, Suzanne; Dube, Simant; Mir, Alain; Qin, Jian; Sun, Gang; Ramakrishnan, Ramesh; Jones, Robert C; Livak, Kenneth J
Reagent decontamination to eliminate false-positives in Escherichia coli qPCR
The application of real-time quantitative PCR (qPCR) for the detection of low concentrations of Escherichia coli as well as universal 16S rDNA has been hindered by false-positives due to endogenous contamination of PCR reagents with E. coli and other bacterial DNA. We optimized a DNase I decontamination method to eliminate false-positives in a qPCR assay targeting the uidA gene in
Sarah S. Silkie; Matthew P. Tolcher; Kara L. Nelson
Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue
Background Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. Methodology/Principal Findings Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n?=?2 and n?=?33. Conclusions/Significance CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue.
Chechi, Kanta; Gelinas, Yves; Mathieu, Patrick; Deshaies, Yves; Richard, Denis
RNA quality assessment: a view from plant qPCR studies.
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is probably the most common molecular technique used in transcriptome analyses today. The simplicity of the technology and associated protocols that generate results without the need to understand the underlying principles has made RT-qPCR the method of choice for RNA quantification. Rather than the 'gold standard technology' often used to describe it, the performance of RT-qPCR suffers from considerable pitfalls during general workflow. The inconsistency of conventional methods for the evaluation of RNA quality and its influence on qPCR performance as well as stability of reference genes is summarized and discussed here. PMID:23045609
Die, Jose V; Román, Belén
Suitable Reference Gene Selection for Different Strains and Developmental Stages of the Carmine Spider Mite, Tetranychus cinnabarinus, using Quantitative Real-Time PCR
Reference genes are used as internal controls in gene expression studies, but their expression levels vary according to tissue types and experimental treatments. Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. In this study, the suitability of eight commonly used genes (??-actin, 5.8SrRNA, ??-TUB, GAPDH, RPL13a, RPS18, TBP, SDHA) were cloned and investigated to find the most stable candidates for normalizing real-time PCR data generated from the four different strains (abamectin-resistant, fenpropathrin-resistant, omethoate-resistant, and susceptible strains) and different developmental stages (eggs, protonymphs, nymphs, and adults) of carmine spider mite, Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae). The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, RPS18 and 5.8SrRNA had the most stable expression regardless of the four different strains, whereas RPS18 and ??-TUB were expressed most stably in different developmental stages.
Sun, W.; Jin, Y.; He, L; Lu, W-C.; Li, M.
Normalizing for biology: accounting for technical and biological variation in levels of reference gene and insulin-like growth factor 1 (igf1) transcripts in fish livers.
Feeding, fasting and re-feeding is a common experimental paradigm for studying growth endocrinology. Herein we demonstrate dynamic changes in the livers of coho salmon under these conditions and how changes in liver composition can influence quantification and interpretation of liver gene expression data. A three-week fast resulted in decreases in hepatosomatic index (liver size), liver glycogen content, and liver DNA concentration. In addition, significant differences were found in liver transcript levels from fed and fasted fish for the reference genes, arp and ef1a, when these were normalized to total RNA. We took the additional step of normalizing reference gene transcript levels to the liver homogenate RNA/DNA ratio to account for differences in RNA yield/cell and the number of cells sampled, normalizing to transcript number per cell rather than transcript number per unit RNA. After this additional step no significant differences in liver transcript levels of reference genes were found. The significance of these results was illustrated by normalizing liver transcript levels of insulin-like growth factor 1 (igf1) to ef1a transcript levels or ef1a transcript levels by RNA/DNA. The different normalization strategies resulted in differing patterns of change in igf1 transcript levels between fed and fasted fish. The novelty of this work rests in a two-step normalization process, attempting to account for both 1) technical errors in reverse transcription and qPCR reactions, and 2) biological variance in liver samples. PMID:22546511
Metzger, David C; Luckenbach, J Adam; Shimizu, Munetaka; Beckman, Brian R
Selection of Reference Gene Expression in a Schizophrenia Brain Cohort
Objective To conduct postmortem human brain research into the neuropathological basis of schizophrenia, it is critical to establish cohorts that are well-characterised and well-matched. Our objective was to determine if specimen characteristics, including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs. Methods We selected a matched cohort of 74 cases (37 schizophrenia / schizoaffective disorder cases and 37 controls cases). Middle frontal gyrus tissue was pulverised, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using RT-PCR, we measured nine housekeeper genes and calculated a geomean in each diagnostic group. Results We found that the RINs were very good (mean 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH, and two clinical variables, agonal state and duration of illness did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. Our results show that people with schizophrenia had significantly less PPIA, and SDHA and tended to have less GUSB and B2M mRNA suggesting that these control genes may not be good candidates for normalisation. Conclusions In our cohort, less than 10% variability in RIN values was detected and the diagnostic groups were well matched overall. Our cohort was adequately powered (0.80–0.90) to detect mRNA differences (25%) due to disease. Our study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.
Weickert, Cynthia Shannon; Sheedy, Donna; Rothmond, Debora A.; Dedova, Irina; Fung, Samantha; Garrick, Therese; Wong, Jenny; Harding, Antony J.; Sivagnanansundaram, Sinthuja; Hunt, Clare; Duncan, Carlotta; Sundqvist, Nina; Tsai, Shan-Yuan; Anand, Jasna; Draganic, Daren; Harper, Clive
Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain
BACKGROUND: Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as \\
Nigel C Noriega; Steven G Kohama; Henryk F Urbanski
Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions
Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its\\u000a success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of\\u000a reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes\\u000a were selected, and
Lei ChenHai-ying; Hai-ying Zhong; Jian-fei Kuang; Jian-guo Li; Wang-jin Lu; Jian-ye Chen
Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.).
Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions. PMID:23933278
Wang, Min; Wang, Qinglian; Zhang, Baohong
Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues
To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.
Cheng, Daifeng; Zhang, Zhiling; He, Xiaofang; Liang, Guangwen
Selection and validation of endogenous reference genes using a high throughput approach
Background Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines. Results Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used. Conclusions This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.
Jin, Ping; Zhao, Yingdong; Ngalame, Yvonne; Panelli, Monica C; Nagorsen, Dirk; Monsurro, Vladia; Smith, Kina; Hu, Nan; Su, Hua; Taylor, Phil R; Marincola, Francesco M; Wang, Ena
Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions
Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best
Valéria Mafra; Karen S. Kubo; Marcio Alves-Ferreira; Marcelo Ribeiro-Alves; Rodrigo M. Stuart; Leonardo P. Boava; Carolina M. Rodrigues; Marcos A. Machado
Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions
Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.
Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen
A total of 82 quantitative PCR (QPCR) assays were used to identify and quantify different indoor molds in dust samples from the homes of six infants suffering from pulmonary hemorrhage and 26 reference homes in Cleveland, Ohio. No significant difference was seen in the total cell...
Suitability of endogenous reference genes for gene expression studies with human intraocular endothelial cells
Background The use of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become widely applied as a method to measure transcript abundance. In order to be reflective of biological processes during health and disease this method is dependent on normalisation of data against stable endogenous controls. However, these genes can vary in their stability in different cell types. The importance of reference gene validation for a particular cell type is now well recognised and is an important step in any gene expression study. Results Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms. Conclusions We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.
Selection of optimal reference genes for normalization in quantitative RT-PCR
Background Normalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes. Results We propose a robust approach for selecting optimal subset(s) of reference genes with the smallest variance of the corresponding normalizing factors. The normalizing factor variance estimates are based on the estimated unstructured covariance matrix of all available candidate reference genes, adjusting for all possible correlations. Robustness is achieved through bootstrapping all candidate reference gene data and obtaining the bootstrap upper confidence limits for the variances of the log-transformed normalizing factors. The selection of the reference gene subset is optimized with respect to one of the following criteria: (A) to minimize the variability of the normalizing factor; (B) to minimize the number of reference genes with acceptable upper limit on variability of the normalizing factor, (C) to minimize the average rank of the variance of the normalizing factor. The proposed approach evaluates all gene subsets of various sizes rather than ranking individual reference genes by their stability, as in the previous work. In two publicly available data sets and one new data set, our approach identified subset(s) of reference genes with smaller empirical variance of the normalizing factor than in subsets identified using previously published methods. A small simulation study indicated an advantage of the proposed approach in terms of sensitivity to identify the true optimal reference subset in the presence of even modest, especially negative correlation among the candidate reference genes. Conclusions The proposed approach performs comprehensive and robust evaluation of the variability of normalizing factors based on all possible subsets of candidate reference genes. The results of this evaluation provide flexibility to choose from important criteria for selecting the optimal subset(s) of reference genes, unless one subset meets all the criteria. This approach identifies gene subset(s) with smaller variability of normalizing factors than current standard approaches, particularly if there is some nontrivial innate correlation among the candidate genes.
Identification of reference genes for normalization of gene expression in thoroughbred and Jeju native horse(Jeju pony) tissues
Quantitative analysis of horse gene expression profiles under diverse experimental conditions is limited by the lack of reliable\\u000a reference genes for normalization of mRNA levels. Therefore, in this study, the expression of potential reference genes was\\u000a compared between thoroughbred and Jeju native horse (Jeju pony). We compared the expression of nine genes by quantitative\\u000a real-time RT-PCR in fourteen tissues between
Kung Ahn; Jin-Han Bae; Kyu-Hwi Nam; Chong-Eon Lee; Kyung-Do Park; Hak-Kyo Lee; Byung-Wook Cho; Heui-Soo Kim
Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR
BACKGROUND: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study
Hyun-Wook Rho; Byoung-Chan Lee; Eun-Seok Choi; Il-Ju Choi; Yeon-Su Lee; Sung-Ho Goh
QPCR: Application for real-time PCR data management and analysis
BACKGROUND: Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several
Stephan Pabinger; Gerhard G. Thallinger; René Snajder; Heiko Eichhorn; Robert Rader; Zlatko Trajanoski
Recent advances in quantitative PCR (qPCR) applications in food microbiology.
Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported. PMID:21569926
Postollec, Florence; Falentin, Hélčne; Pavan, Sonia; Combrisson, Jérôme; Sohier, Daničle
Real-Time PCR (qPCR) Primer Design Using Free Online Software
ERIC Educational Resources Information Center
|Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…
Thornton, Brenda; Basu, Chhandak
Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress. PMID:22347455
Mafra, Valéria; Kubo, Karen S; Alves-Ferreira, Marcio; Ribeiro-Alves, Marcelo; Stuart, Rodrigo M; Boava, Leonardo P; Rodrigues, Carolina M; Machado, Marcos A
Evaluation of Candidate Reference Genes for Gene Expression Normalization in Brassica juncea Using Real Time Quantitative RT-PCR
The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.
Chandna, Ruby; Augustine, Rehna; Bisht, Naveen C.
Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR.
Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-?, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR. PMID:23508400
Yin, Zhiyuan; Ke, Xiwang; Huang, Dingxuan; Gao, Xiaoning; Voegele, Ralf T; Kang, Zhensheng; Huang, Lili
Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development
Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares – geNorm, NormFinder and BestKeeper – were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1? (elongation factor 1-?) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1? (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes.
Han, Xiaojiao; Lu, Mengzhu; Chen, Yicun; Zhan, Zhiyong; Cui, Qinqin; Wang, Yangdong
Evaluation of reference genes for expression studies in ash ...
Source: Plant Molecular Biology Reporter. ... Real-time quantitative polymerase chain reaction (RTq-PCR) is widely used for validating transcript levels in gene expression ... During the capture process some typographical errors may occur.
AutDB: a gene reference resource for autism research
Recent advances in studies of Autism Spectrum Disorders (ASD) has uncovered many new candidate genes and continues to do so at an accelerated pace. To address the genetic complexity of ASD, we have developed AutDB (http:\\/\\/www.mindspec. org\\/autdb.html), a publicly available web-portal for on-going collection, manual annotation and visuali- zation of genes linked to the disorder. We present a disease-driven database
Saumyendra N. Basu; Ravi Kollu; Sharmila Banerjee-basu
Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA
Background As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P?0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. Conclusion There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.
Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration.
It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell types, developmental stages, and/or sample treatment. To select the most stable endogenous reference genes during planarian regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder software. Expression levels of the Djsix-1 gene were studied in parallel with ACTB and DjEF2 both or each and 18S rRNA as reference during regeneration. The results showed that ACTB was the most stable expressed reference gene in the planarian regeneration. PMID:21161407
Yuwen, Yan-Qing; Dong, Zi-Mei; Wang, Qing-Hua; Sun, Xiao-Juan; Shi, Chang-Ying; Chen, Guang-Wen
Characterization of reference gene expression in tung tree (Vernicia fordii)
Technology Transfer Automated Retrieval System (TEKTRAN)
Tung oil from tung tree (Vernicia fordii) is widely used as a drying ingredient in paints, varnishes, and other coatings and finishes. Recent research has focused on the understanding of the biosynthesis of oil in tung trees. Many oil biosynthetic genes have been identified in tung tree but little...
Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis
Background Bivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and ?-actin (ACT) is most frequently used as qRT-PCR reference gene without validation. Results In this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopecten yessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ?Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae. Conclusions This work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.
Feng, Liying; Yu, Qian; Li, Xue; Ning, Xianhui; Wang, Jing; Zou, Jiajun; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli; Bao, Zhenmin
Selection of Reference Genes for Quantitative Real-Time PCR in Bamboo (Phyllostachys edulis)
Background The Moso bamboo (Phyllostachys edulis) is one of the most important forestry resources and plays essential ecological roles in southern China. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression data related to the unique traits of Moso bamboo will undoubtedly follow. Reverse transcription quantitative real-time PCR ((RT-)qPCR) is a widely used method for gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. Result In this study, 14 candidate reference genes were chosen, and their expression levels were assessed by (RT-)qPCR in a set of six tissue samples (root, stem, mature stem, leaf, flower, and leaf sheath) and at two developmental stages (before and after flowering) in bamboo specimens obtained in three locations. The stability and suitability of the candidate reference genes were validated using the geNorm, NormFinder and BestKeeper programs. The results showed that TIP41 and NTB were suitable reference genes across all the tissues and at the different developmental stages examined in this study. While the expression of the NTB, TIP41 and UBQ were the mostly stable in different plant tissues samples, the expression of the TIP41, NTB and CAC were ranked the most stable in bamboo plants at various developmental stages. AP2-like gene was further assessed by using the reference genes TIP41 and NTB in comparison to ACT. Significant difference of the expression profile of AP2-like demonstrated the importance of choosing adequate reference genes in bamboo. Conclusion TIP41 and NTB were found to be homogeneously expressed and were adequate for normalization purposes, showing equivalent transcript levels in different samples. They are therefore the recommended reference genes for measuring gene expression in P. edulis.
Fan, Chunjie; Ma, Jinmin; Guo, Qirong; Li, Xiaotie; Wang, Hui; Lu, Mengzhu
Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain
Background Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods. We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix® GeneChip® rhesus Macaque Genome Array. Having shown that qRT-PCR and Affymetrix® GeneChip® data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages. Results Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip® facilitated more targeted analysis than could be accomplished using the rhesus GeneChip®. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed. Conclusions Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.
Leukocyte count affects expression of reference genes in canine whole blood samples
Background The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated. Findings The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that underwent a surgical procedure. GeNorm analysis revealed that a combination of 5 to 6 of the most stably expressed genes constituted a stable normalizing factor. Evaluation of the expression revealed different ranking of reference genes in Normfinder and GeNorm. The disease category and the white blood cell count significantly affected reference gene expression. Conclusions The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count". This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.
New in-depth rainbow trout transcriptome reference and digital atlas of gene expression
Technology Transfer Automated Retrieval System (TEKTRAN)
Sequencing the rainbow trout genome is underway and a transcriptome reference sequence is required to help in genome assembly and gene discovery. Previously, we reported a transcriptome reference sequence using a 19X coverage of 454-pyrosequencing data. Although this work added a great wealth of ann...
An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry.
Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry. PMID:21761237
Vashisth, Tripti; Johnson, Lisa Klima; Malladi, Anish
Revealing the missing expressed genes beyond the human reference genome by RNA-Seq
Background The complete and accurate human reference genome is important for functional genomics researches. Therefore, the incomplete reference genome and individual specific sequences have significant effects on various studies. Results we used two RNA-Seq datasets from human brain tissues and 10 mixed cell lines to investigate the completeness of human reference genome. First, we demonstrated that in previously identified ~5 Mb Asian and ~5 Mb African novel sequences that are absent from the human reference genome of NCBI build 36, ~211 kb and ~201 kb of them could be transcribed, respectively. Our results suggest that many of those transcribed regions are not specific to Asian and African, but also present in Caucasian. Then, we found that the expressions of 104 RefSeq genes that are unalignable to NCBI build 37 in brain and cell lines are higher than 0.1 RPKM. 55 of them are conserved across human, chimpanzee and macaque, suggesting that there are still a significant number of functional human genes absent from the human reference genome. Moreover, we identified hundreds of novel transcript contigs that cannot be aligned to NCBI build 37, RefSeq genes and EST sequences. Some of those novel transcript contigs are also conserved among human, chimpanzee and macaque. By positioning those contigs onto the human genome, we identified several large deletions in the reference genome. Several conserved novel transcript contigs were further validated by RT-PCR. Conclusion Our findings demonstrate that a significant number of genes are still absent from the incomplete human reference genome, highlighting the importance of further refining the human reference genome and curating those missing genes. Our study also shows the importance of de novo transcriptome assembly. The comparative approach between reference genome and other related human genomes based on the transcriptome provides an alternative way to refine the human reference genome.
RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization
Background RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements\\u000a between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however,\\u000a that the expression of commonly used reference genes can vary significantly in certain contexts.\\u000a \\u000a \\u000a \\u000a \\u000a Results Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression
Tomas Hruz; Markus Wyss; Mylene Docquier; Michael W Pfaffl; Sabine Masanetz; Lorenzo Borghi; Phebe Verbrugghe; Luba Kalaydjieva; Stefan Bleuler; Oliver Laule; Patrick Descombes; Wilhelm Gruissem; Philip Zimmermann
Immunome: A reference set of genes and proteins for systems biology of the human immune system
The function of the immune system requires coordinated expression and action of many genes and proteins. With the emergence of high throughput technologies it has become possible to target human immunity from a systems biology point of view. To perform systems biology studies with comparable results we need not just a reference gene and protein set but also useful and
Csaba Ortutay; Mauno Vihinen
Reference Gene Selection for Quantitative Real-time PCR Normalization in Quercus suber
The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1?, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.
Marum, Liliana; Miguel, Andreia; Ricardo, Candido P.; Miguel, Celia
Tuber aestivum Vittad. mycelium quantified: advantages and limitations of a qPCR approach.
A quantitative real-time PCR (qPCR) marker Ta0 with hydrolysis probe ("TaqMan"), targeted to the internal transcribed spacer region of the ribosomal DNA, has been developed for quantification of summer truffle (Tuber aestivum) mycelium. Gene copy concentrations determined by the qPCR were calibrated against pure culture mycelium of T. aestivum, enabling quantification of the mycelium in soil and in host roots from the fields. Significant concentrations of the fungus were observed not only in the finest roots with ectomycorrhizae but also in other root types, indicating that the fungus is an important component of the microbial film at the root surface. The concentration of T. aestivum in soil is relatively high compared to other ectomycorrhizal fungi. To evaluate the reliability of the measurement of the soil mycelium density using qPCR, the steady basal extracellular concentration of the stabilized T. aestivum DNA should be known and taken into account. Therefore, we addressed the stability of the qPCR signal in soil subjected to different treatments. After the field soil was sieved, regardless of whether it was dried/rewetted or not, the T. aestivum DNA was quickly decomposed. It took just about 4 days to reach a steady concentration. This represents a conserved pool of T. aestivum DNA and determines detection limit of the qPCR quantification in our case. When the soil was autoclaved and recolonized by saprotrophic microorganisms, this conserved DNA pool was eliminated and the soil became free of T. aestivum DNA. PMID:23271632
Gryndler, Milan; Tril?ová, Jana; Hršelová, Hana; Streiblová, Eva; Gryndlerová, Hana; Jansa, Jan
Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae).
Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ?Ct method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms. PMID:23874494
Lu, Yanhui; Yuan, Miao; Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong
Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria.
Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works. PMID:22496882
Pinto, Filipe; Pacheco, Catarina C; Ferreira, Daniela; Moradas-Ferreira, Pedro; Tamagnini, Paula
Impact of thawing on reference gene expression stability in renal cell carcinoma samples.
More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M=0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M=1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P<0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P<0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods. PMID:22847160
Ma, Yi; Dai, HuiLi; Kong, XianMing; Wang, LiMin
Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis
Reverse transcription and real-time PCR (RT-qPCR) has been widely used for rapid quantification of relative gene expression. To offset technical confounding variations, stably-expressed internal reference genes are measured simultaneously along with target genes for data normalization. Statistic methods have been developed for reference validation; however normalization of RT-qPCR data still remains arbitrary due to pre-experimental determination of particular reference genes.
Daijun Ling; Paul M. Salvaterra; Baochuan Lin
Detection of Streptococcus equi subspecies equi using a triplex qPCR assay.
Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria. PMID:22884566
Webb, Katy; Barker, Colin; Harrison, Tihana; Heather, Zoe; Steward, Karen F; Robinson, Carl; Newton, J Richard; Waller, Andrew S
Method A: Enterococci in Water by TaqMan (Trade Name) Quantitative Polymerase Chain Reaction (qPCR) Assay.
National Technical Information Service (NTIS)
Method A describes a quantitative polymerase chain reaction (qPCR) procedure for the detection of DNA from enterococci bacteria in ambient water matrices based on the amplification and detection of a specific region of the large subunit ribosomal RNA gene...
Selection of endogenous reference genes for gene expression analysis in the Mediterranean species of the Bemisia tabaci (Hemiptera: Aleyrodidae) complex.
Quantitative real-time reverse transcription polymerase chain reaction is widely used for gene expression analysis, and robust normalization against stably expressed endogenous reference genes (ERGs) is necessary to obtain accurate results. In this study, the stability of nine housekeeping genes of the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) Mediterranean were evaluated in various conditions by quantitative real-time reverse transcription polymerase chain reaction using geNorm and Normfinder programs. Both programs suggested alpha-tubulin/ubiquitin and 18S small subunit ribosomal RNA the most stable genes for bacterium- and insecticide-treated whiteflies, respectively. For developmental stages, organs, and the samples including salivary glands and the whole body, transcription initiation factor TFIID subunit was calculated as the most stably expressed gene by both programs. In addition, we compared the RNA-seq data with the results of geNorm and Normfinder and found that the stable genes revealed by RNA-seq analysis were also the ERGs recommended by geNorm and Normfinder. Furthermore, the use of the most stable gene suggested by RNA-seq analysis as an ERG produced similar gene expression patterns compared with results generated from the normalization against the most stable gene selected by geNorm and Normfinder and multiple genes recommended by geNorm. It indicates that RNA-seq data are reliable and provide a great source for ERG candidate exploration. Our results benefit future research on gene expression profiles of whiteflies and possibly other organisms. PMID:23865213
Su, Yun; He, Wen-Bo; Wang, Jia; Li, Jun-Min; Liu, Shu-Sheng; Wang, Xiao-Wei
Selection and Validation of Reference Genes for miRNA Expression Studies during Porcine Pregnancy
MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.
Wessels, Jocelyn M.; Edwards, Andrew K.; Zettler, Candace; Tayade, Chandrakant
Guideline to reference gene selection for quantitative real-time PCR
Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and ?-actin have been used for that purpose. However, it has been reported that these genes as
Aleksandar Radoni?; Stefanie Thulke; Ian M. Mackay; Olfert Landt; Wolfgang Siegert; Andreas Nitschea
Identification of Mouse Serum miRNA Endogenous References by Global Gene Expression Profiles
MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female
Qing-Sheng Mi; Matthew Weiland; Rui-Qun Qi; Xing-Hua Gao; Laila M. Poisson; Li Zhou
A universal reference sample derived from clone vector for improved detection of differential gene expression
Background Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs™. Results We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. Conclusion Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes.
Khan, Rishi L; Gonye, Gregory E; Gao, Guang; Schwaber, James S
The 'PREXCEL-Q Method' for qPCR
The purpose of this manuscript is to describe a reliable approach to quantitative real-time polymerase chain reaction (qPCR) assay development and project management, which is currently embodied in the Excel 2003-based software program named "PREXCEL-Q" (P-Q) (formerly known as "FocusField2-6Gallup-qPCRSet-upTool-001," "FF2-6-001 qPCR set-up tool" or "Iowa State University Research Foundation [ISURF] project #03407"). Since its inception from 1997-2007, the program has been well-received and requested around the world and was recently unveiled by its inventor at the 2008 Cambridge Healthtech Institute's Fourth Annual qPCR Conference in San Diego, CA. P-Q was subsequently mentioned in a review article by Stephen A. Bustin, an acknowledged leader in the qPCR field. Due to its success and growing popularity, and the fact that P-Q introduces a unique/defined approach to qPCR, a concise description of what the program is and what it does has become important. Sample-related inhibitory problems of the qPCR assay, sample concentration limitations, nuclease-treatment, reverse transcription (RT) and master mix formulations are all addressed by the program, enabling investigators to quickly, consistently and confidently design uninhibited, dynamically-sound, LOG-linear-amplification-capable, high-efficiency-of-amplification reactions for any type of qPCR. The current version of the program can handle an infinite number of samples.
Gallup, Jack M.; Ackermann, Mark R.
A Brain RegionSpecific Predictive Gene Map for Autism Derived by Profiling a Reference Gene Set
Molecular underpinnings of complex psychiatric disorders such as autism spectrum disorders (ASD) remain largely unresolved. Increasingly, structural variations in discrete chromosomal loci are implicated in ASD, expanding the search space for its disease etiology. We exploited the high genetic heterogeneity of ASD to derive a predictive map of candidate genes by an integrated bioinformatics approach. Using a reference set of
Ajay Kumar; Catherine Croft Swanwick; Nicole Johnson; Idan Menashe; Saumyendra N. Basu; Michael E. Bales; Sharmila Banerjee-Basu
Unification of gene expression data applying standard mRNA quantification references for comparable analyses
Technology Transfer Automated Retrieval System (TEKTRAN)
High throughput quantitative measurements of gene expression data have problems of reproducibility and comparability due to a lack of standard mRNA quantification references. Efforts have been made to safeguard data fidelity, yet generating quality expression data of inherent value remains a challe...
Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR.
The genetically modified (GM) food/feed quantification depends on the reliable detection systems of endogenous reference genes. Currently, four endogenous reference genes including sucrose phosphate synthase (SPS), GOS9, phospholipase D (PLD), and ppi phosphofructokinase (ppi-PPF) of rice have been used in GM rice detection. To compare the applicability of these four rice reference genes in quantitative PCR systems, we analyzed the target nucleotide sequence variation in 58 conventional rice varieties from various geographic and phylogenic origins, also their quantification performances were evaluated using quantitative real-time PCR and GeNorm analysis via a series of statistical calculation to get a "M value" which is negative correlation with the stability of genes. The sequencing analysis results showed that the reported GOS9 and PLD taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested rice cultivars, while no SNPs were observed for SPS and ppi-PPF amplicons. Also, poor quantitative performance was detectable in these cultivars with SNPs using GOS9 and PLD quantitative PCR systems. Even though the PCR efficiency of ppi-PPF system was slightly lower, the SPS and ppi-PPF quantitative PCR systems were shown to be applicable for rice endogenous reference assay with less variation among the C(t) values, good reproducibility in quantitative assays, and the low M values by the comprehensive quantitative PCR comparison and GeNorm analysis. PMID:20961039
Wang, Chong; Jiang, Lingxi; Rao, Jun; Liu, Yinan; Yang, Litao; Zhang, Dabing
A Novel Strategy for Selection and Validation of Reference Genes in Dynamic Multidimensional Experimental Design in Yeast
Background Understanding the dynamic mechanism behind the transcriptional organization of genes in response to varying environmental conditions requires time-dependent data. The dynamic transcriptional response obtained by real-time RT-qPCR experiments could only be correctly interpreted if suitable reference genes are used in the analysis. The lack of available studies on the identification of candidate reference genes in dynamic gene expression studies necessitates the identification and the verification of a suitable gene set for the analysis of transient gene expression response. Principal Findings In this study, a candidate reference gene set for RT-qPCR analysis of dynamic transcriptional changes in Saccharomyces cerevisiae was determined using 31 different publicly available time series transcriptome datasets. Ten of the twelve candidates (TPI1, FBA1, CCW12, CDC19, ADH1, PGK1, GCN4, PDC1, RPS26A and ARF1) we identified were not previously reported as potential reference genes. Our method also identified the commonly used reference genes ACT1 and TDH3. The most stable reference genes from this pool were determined as TPI1, FBA1, CDC19 and ACT1 in response to a perturbation in the amount of available glucose and as FBA1, TDH3, CCW12 and ACT1 in response to a perturbation in the amount of available ammonium. The use of these newly proposed gene sets outperformed the use of common reference genes in the determination of dynamic transcriptional response of the target genes, HAP4 and MEP2, in response to relaxation from glucose and ammonium limitations, respectively. Conclusions A candidate reference gene set to be used in dynamic real-time RT-qPCR expression profiling in yeast was proposed for the first time in the present study. Suitable pools of stable reference genes to be used under different experimental conditions could be selected from this candidate set in order to successfully determine the expression profiles for the genes of interest.
Cankorur-Cetinkaya, Ayca; Dereli, Elif; Eraslan, Serpil; Karabekmez, Erkan; Dikicioglu, Duygu; Kirdar, Betul
Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L.).
Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technology for gene expression and transcriptome analysis. Normalization is a process that is necessary to accurately analyze qRT-PCR data. Stability of reference gene expression is required for this process. Due to the large variation in expression levels of reference genes obtained from different experimental conditions, gene expression stabilities must be evaluated and identified in all experimental systems. In the present paper, the stability of the expression levels of seven potential reference genes in pepper are assessed using qRT-PCR analysis to determine optimal reference genes. These reference genes are evaluated in different pepper tissues, abiotic stress, and hormonal treatment samples. Three common statistical algorithms, geNorm, NormFinder, and BestKeeper, are used to identify expression stability and provide an accurate selection of reference genes. Two reference genes, beta tubulin and ubiquitin-conjugating protein (UBI-3), showed high stability in sample pools with abiotic stress and hormonal treatments. Among the sample pools tested, UBI-3 and glyceraldehyde-3-phosphate dehydrogenase expression levels were the most stable in different tissues. Therefore, these reference genes are selected for qRT-PCR analysis under the experimental conditions tested in pepper. In contrast, ubiquitin-conjugating enzyme and actin genes are identified as the least stable reference genes in all the groups tested, confirming that they are not suitable for normalization. Validation of these candidate genes could provide useful guidelines for reference gene selection in qRT-PCR studies in pepper. PMID:22086175
Wan, Hongjian; Yuan, Wei; Ruan, Meiying; Ye, Qingjing; Wang, Rongqing; Li, Zhimiao; Zhou, Guozhi; Yao, Zhuping; Zhao, Jing; Liu, Shujun; Yang, Yuejian
Sampling Daphnia's expressed genes: preservation, expansion and invention of crustacean genes with reference to insect genomes
BACKGROUND: Functional and comparative studies of insect genomes have shed light on the complement of genes, which in part, account for shared morphologies, developmental programs and life-histories. Contrasting the gene inventories of insects to those of the nematodes provides insight into the genomic changes responsible for their diversification. However, nematodes have weak relationships to insects, as each belongs to separate
John K Colbourne; Brian D Eads; Joseph Shaw; Elizabeth Bohuski; Darren J Bauer; Justen Andrews
Systematic validation of candidate reference genes for qRT-PCR normalization under iron deficiency in Arabidopsis.
A reliable result obtained by qRT-PCR highly depends on accurate transcript normalization using stably expressed reference genes. However, the transcript levels of traditional reference genes are not always stable. Also, the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated in Arabidopsis under iron deficiency. Our results revealed that three novel reference genes (SAND, YLS8 and TIP41-like) were identified and validated as suitable reference genes for qRT-PCR normalization in both iron deprivation (the addition of Ferrozine to the medium) and starvation (withdrawal of iron from the medium) conditions. This conclusion was also confirmed by publicly available microarray data. In addition, when using SAND, YLS8 and TIP41-like as multiple reference genes, the expression patterns of FIT1 and IRT1, two iron deficiency marker genes, were approximately similar with that reported previously. However, a weaker inducible response was obtained from qRT-PCR by normalizating EF-1? alone. Together, we proposed that the combination of SAND, YLS8 and TIP41-like can be used for accurate normalization of gene expression in iron deficiency research. These results provide a valuable evidence for the importance of adequate reference genes in qRT-PCR normalization, insisting on the use of appropriate reference gene validation in all transcriptional analyses. PMID:23547009
Han, Bin; Yang, Zheng; Samma, Muhammad Kaleem; Wang, Ren; Shen, Wenbiao
Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.
Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius. PMID:21845960
Mamidala, Praveen; Rajarapu, Swapna P; Jones, Susan C; Mittapalli, Omprakash
Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae
BACKGROUND: Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions.
Marie-Ange Teste; Manon Duquenne; Jean M François; Jean-Luc Parrou
Sampling Daphnia's expressed genes: preservation, expansion and invention of crustacean genes with reference to insect genomes
Background Functional and comparative studies of insect genomes have shed light on the complement of genes, which in part, account for shared morphologies, developmental programs and life-histories. Contrasting the gene inventories of insects to those of the nematodes provides insight into the genomic changes responsible for their diversification. However, nematodes have weak relationships to insects, as each belongs to separate animal phyla. A better outgroup to distinguish lineage specific novelties would include other members of Arthropoda. For example, crustaceans are close allies to the insects (together forming Pancrustacea) and their fascinating aquatic lifestyle provides an important comparison for understanding the genetic basis of adaptations to life on land versus life in water. Results This study reports on the first characterization of cDNA libraries and sequences for the model crustacean Daphnia pulex. We analyzed 1,546 ESTs of which 1,414 represent approximately 787 nuclear genes, by measuring their sequence similarities with insect and nematode proteomes. The provisional annotation of genes is supported by expression data from microarray studies described in companion papers. Loci expected to be shared between crustaceans and insects because of their mutual biological features are identified, including genes for reproduction, regulation and cellular processes. We identify genes that are likely derived within Pancrustacea or lost within the nematodes. Moreover, lineage specific gene family expansions are identified, which suggest certain biological demands associated with their ecological setting. In particular, up to seven distinct ferritin loci are found in Daphnia compared to three in most insects. Finally, a substantial fraction of the sampled gene transcripts shares no sequence similarity with those from other arthropods. Genes functioning during development and reproduction are comparatively well conserved between crustaceans and insects. By contrast, genes that were responsive to environmental conditions (metal stress) and not sex-biased included the greatest proportion of genes with no matches to insect proteomes. Conclusion This study along with associated microarray experiments are the initial steps in a coordinated effort by the Daphnia Genomics Consortium to build the necessary genomic platform needed to discover genes that account for the phenotypic diversity within the genus and to gain new insights into crustacean biology. This effort will soon include the first crustacean genome sequence.
Colbourne, John K; Eads, Brian D; Shaw, Joseph; Bohuski, Elizabeth; Bauer, Darren J; Andrews, Justen
Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa.
Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for non-model plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes should be thoroughly investigated for each experimental condition under investigation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-012-9711-1) contains supplementary material, which is available to authorized users. PMID:23024595
Podevin, Nancy; Krauss, An; Henry, Isabelle; Swennen, Rony; Remy, Serge
Quantification of Gene Expression after Painful Nerve Injury: Validation of Optimal Reference Genes
Stably expressed housekeeping genes (HKGs) are necessary for standardization of transcript measurement by quantitative real-time\\u000a polymerase chain reaction (qRT-PCR). Peripheral nerve injury disrupts expression of numerous genes in sensory neurons, but\\u000a the stability of conventional HKGs has not been tested in this context. We examined the stability of candidate HKGs during\\u000a nerve injury, including the commonly used 18S ribosomal RNA,
Madhavi Latha Yadav Bangaru; Frank Park; Andy Hudmon; J. Bruce McCallum; Quinn H. Hogan
Appropriate reference gene selection for real-time PCR data normalization during rat mesenchymal stem cell differentiation.
Reverse transcription quantitative PCR (RT—qPCR) is one of the best methods for the study of mesenchymal stem cell (MSC) differentiation by gene expression analysis. This technique needs appropriate reference or housekeeping genes (HKGs) to normalize the expression of the genes of interest. In the present study the expression stability of six widely used HKGs including Actb, Btub, Hprt, B2m, Gusb and Tfrc was investigated during rat MSC differentiation into osteocytes, adipocytes and chondrocytes lineages using geNorm and NormFinder software. RT—qPCR data analyzed by geNorm revealed the different sets of suitable reference genes for each cell type. NormFinder also showed similar results. Analysis of the combined data of MSCs with each differentiated cell type revealed the considerable shift in expression of some reference genes during differentiation; for example Gusb and B2m were among the least stable genes in MSCs but the most stable in chondrocytes. Normalization of specific genes for each lineage by different reference genes showed considerable difference in their expression fold change. In conclusion, for the appropriate analysis of gene expression during rat MSC differentiation and also for monitoring differentiation procedures, it is better to consider precisely the reference gene stability and select suitable reference genes for each purpose. PMID:22595340
Farrokhi, A; Eslaminejad, M B; Nazarian, H; Moradmand, A; Samadian, A; Akhlaghi, A
Comparison of five endogenous reference genes for specific PCR detection and quantification of Brassica napus.
Five previously reported Brassica napus endogenous reference genes, including acetyl-CoA carboxylase gene (BnACCg8), phosphoenolpyruvate carboxylase (PEP), oleoyl hydrolase gene (FatA), high-mobility-group protein I/Y gene (HMG-I/Y) and cruciferin A gene (CruA), were analyzed for their PCR specificity between B. napus and other species and the quantification stability among different B. napus cultivars. PCR and sequencing results indicated that none of these systems was species-specific as required by the genetically modified organism labeling policy. When these genes were employed in real-time PCR, BnACCg8 and HMG-I/Y systems showed relatively greater heterogeneity among 10 different cultivars. The sequencing results showed that the single nucleotide polymorphism in the primer binding sites was the potential source of the instability in the HMG-I/Y system. The bias of BnACCg8 was thought to be associated with the inconsistent copy number of this gene. PMID:20143854
Wu, Gang; Zhang, Li; Wu, Yuhua; Cao, Yinglong; Lu, Changming
Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-? - a method for qRT-PCR reference gene determination
Based on the exquisite sensitivity, reproducibility, and wide dynamic range of quantitative reverse-transcription real-time polymerase-chain-reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable "best" reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-? was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan® qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8, and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-?-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes.
Riemer, Angelika B.; Keskin, Derin B.; Reinherz, Ellis L.
Identification of Novel Reference Genes Using Multiplatform Expression Data and Their Validation for Quantitative Gene Expression Analysis
Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required.
Kwon, Mi Jeong; Oh, Ensel; Lee, Seungmook; Roh, Mi Ra; Kim, Si Eun; Lee, Yangsoon; Choi, Yoon-La; In, Yong-Ho; Park, Taesung; Koh, Sang Seok; Shin, Young Kee
Identification of novel reference genes using multiplatform expression data and their validation for quantitative gene expression analysis.
Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required. PMID:19584937
Kwon, Mi Jeong; Oh, Ensel; Lee, Seungmook; Roh, Mi Ra; Kim, Si Eun; Lee, Yangsoon; Choi, Yoon-La; In, Yong-Ho; Park, Taesung; Koh, Sang Seok; Shin, Young Kee
SYBR ® Green qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of genetically modified crops in food and feed products
Identification of crops present in food and\\/or feed matrices represents an important step in the screening strategies targeting\\u000a genetically modified organisms (GMO). Soybean, maize, oilseed rape, rice, cotton, sugar beet and potato are to date the most\\u000a important sources of genetically modified materials imported in the European Union (EU). In order to allow detection of their\\u000a presence in an integrated
E. Guillaume Mbongolo Mbella; Antoon Lievens; Elodie Barbau-Piednoir; Myriam Sneyers; Amaya Leunda-Casi; Nancy Roosens; Marc Van den Bulcke
Selection of stable reference genes in heat stressed peripheral blood mononuclear cells of tropically adapted Indian cattle and buffaloes.
Eleven reference genes from different functional categories were assessed for their stable expression pattern in heat stressed PBMCs across Indian buffalo and cattle using GeNorm, NormFinder and BestKeeper algorithms. As the first report, we suggest B2M, RPS9 and RPS15a as suitable reference genes for accurate normalization of PBMC transcript data while the ACTB gene is not recommended. PMID:23499765
Kishore, Amit; Sodhi, Monika; Khate, Keviletsu; Kapila, Neha; Kumari, Parvesh; Mukesh, Manishi
Selection of New Appropriate Reference Genes for RT-qPCR Analysis via Transcriptome Sequencing of Cynomolgus Monkeys (Macaca fascicularis)
In the investigation of the expression levels of target genes, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most accurate and widely used method. However, a normalization step is a prerequisite to obtain accurate quantification results from RT-qPCR data. Therefore, many studies regarding the selection of reference genes have been carried out. Recently, these studies have involved large-scale gene analysis methods such as microarray and next generation sequencing. In our previous studies, we analyzed large amounts of transcriptome data from the cynomolgus monkey. Using a modification of this large-scale transcriptome sequencing dataset, we selected and compared 12 novel candidate reference genes (ARFGAP2, ARL1, BMI1, CASC3, DDX3X, MRFAP1, ORMDL1, RSL24D1, SAR1A, USP22, ZC3H11A, and ZRANB2) and 4 traditionally used reference genes (ACTB, GAPDH, RPS19, and YWHAZ) in 13 different whole-body tissues by the 3 well-known programs geNorm, NormFinder, and BestKeeper. Combined analysis by these 3 programs showed that ADP-ribosylation factor GTPase activating protein 2 (ARFGAP2), morf4 family associated protein 1 (MRFAP1), and ADP-ribosylation factor-like 1 (ARL1) are the most appropriate reference genes for accurate normalization. Interestingly, 4 traditionally used reference genes were the least stably expressed in this study. For this reason, selection of appropriate reference genes is vitally important, and large-scale analysis is a good method for finding new candidate reference genes. Our results could provide reliable reference gene lists for future studies on the expression of various target genes in the cynomolgus monkey.
Lee, Sang-Rae; Kim, Sang-Hyun; Kim, Sun-Uk; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Kyoung-Min; Kim, Heui-Soo; Chang, Kyu-Tae
Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR.
Gene expression profiling by quantitative real-time PCR (RT-qPCR) is a valuable tool in forensic science for estimating the age of a wound. To accurately assess gene expression levels over time in injured tissue, the genes used as internal reference standards must be carefully validated for transcriptional stability. This study examined the transcriptional stability of nine potential reference genes (?-actin, GAPDH, RPL32, PGK1, SDHA, RPL13, HPRT, Tbp, and Ywhaz) in contused rat skeletal muscle by RT-qPCR. The raw Ct values were determined for each candidate gene at different time points following contusion, and the data were analyzed by the NormFinder, geNorm, and BestKeeper validation programs. The reference genes RPL13 and RPL32 were the most stably expressed genes in contused skeletal muscle, whereas PGK1 was the least stable. The commonly used reference genes ?-actin and GAPDH appeared to be too unstable for normalization of RT-qPCR expression profiling in contused muscle. The reference genes RPL13 and RPL32 were also the best combination for multianalysis. The use of RPL13 and RPL32 as internal standards may improve the accuracy of gene expression studies aimed at determining the age of early wounds in forensic investigations. PMID:21845441
Sun, Jun-hong; Nan, Liang-huan; Gao, Cai-rong; Wang, Ying-yuan
Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86
BACKGROUND: For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually ?-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference
Ard M Nijhof; Jesper A Balk; Milagros Postigo; Frans Jongejan
Selection of Reliable Reference Genes in Caenorhabditis elegans for Analysis of Nanotoxicity
Despite rapid development and application of a wide range of manufactured metal oxide nanoparticles (NPs), the understanding of potential risks of using NPs is less completed, especially at the molecular level. The nematode Caenorhabditis elegans (C.elegans) has been emerging as an environmental model to study the molecular mechanism of environmental contaminations, using standard genetic tools such as the real-time quantitative PCR (RT-qPCR). The most important factor that may affect the accuracy of RT-qPCR is to choose appropriate genes for normalization. In this study, we selected 13 reference gene candidates (act-1, cdc-42, pmp-3, eif-3.C, actin, act-2, csq-1, Y45F10D.4, tba-1, mdh-1, ama-1, F35G12.2, and rbd-1) to test their expression stability under different doses of nano-copper oxide (CuO 0, 1, 10, and 50 µg/mL) using RT-qPCR. Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ?Ct method, were employed to evaluate these 13 candidates expressions. As a result, tba-1, Y45F10D.4 and pmp-3 were the most reliable, which may be used as reference genes in future study of nanoparticle-induced genetic response using C.elegans.
Zhang, Yanqiong; Chen, Dongliang; Smith, Michael A.; Zhang, Baohong; Pan, Xiaoping
Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae
Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.
Teste, Marie-Ange; Duquenne, Manon; Francois, Jean M; Parrou, Jean-Luc
Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells
Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-?. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that ?-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.
Zyzynska-Granica, Barbara; Koziak, Katarzyna
Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR.
Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis. PMID:22732401
Cao, Shaona; Zhang, Xiaowen; Ye, Naihao; Fan, Xiao; Mou, Shanli; Xu, Dong; Liang, Chengwei; Wang, Yitao; Wang, Wenqi
The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress.
Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1? for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1? and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis. PMID:23979536
Janská, Anna; Hodek, Jan; Svoboda, Pavel; Záme?ník, Ji?í; Prášil, Ilja Tom; Vlasáková, Eva; Milella, Luigi; Ovesná, Jaroslava
Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution
This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies,
Suzanne Weaver; Simant Dube; Alain Mir; Jian Qin; Gang Sun; Ramesh Ramakrishnan; Robert C. Jones; Kenneth J. Livak
Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida
Background Identification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition tested as well as the methods used for RNA purification and analysis. Results In an effort to identify endogenous genes meeting these criteria nine reference genes (RG) were tested in two Petunia lines (Mitchell and V30). Growth conditions differed in Mitchell and V30, and different methods were used for RNA isolation and analysis. Four different software tools were employed to analyze the data. We merged the four outputs by means of a non-weighted unsupervised rank aggregation method. The genes identified as optimal for transcriptomic analysis of Mitchell and V30 were EF1? in Mitchell and CYP in V30, whereas the least suitable gene was GAPDH in both lines. Conclusions The least adequate gene turned out to be GAPDH indicating that it should be rejected as reference gene in Petunia. The absence of correspondence of the best-suited genes suggests that assessing reference gene stability is needed when performing normalization of data from transcriptomic analysis of flower and leaf development.
Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.
Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao
Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection.
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735
Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao
Evaluation of suitable reference genes for normalization of real-time reverse transcription PCR analysis in colon cancer
Background Real-time reverse transcription PCR (qRT-PCR) is frequently used for gene expression quantification due to its methodological reproducibility and sensitivity. The gene expression is quantified by normalization to one or more reference genes which are presumed stably expressed throughout a given experiment. The aim of this study was to validate a standardized experimental setup to identifying reference genes for normalization of qRT-PCR in the metastatic and non-metastatic colon cancer. Methods In this study, expression of 16 commonly used reference genes was quantified in tumour tissue and individual-matched normal mucosa in 18 non-metastatic colon cancer patients and 20 colon cancer patients with distant metastases using TaqMan Low Density Array (TLDA). The expression stability was determined and compared by means of geNorm and NormFinder. Results Two pairs of genes, HPRT1/PPIA and IPO8/PPIA, were identified to be suitable to normalize gene expression data in metastatic and non-metastatic colon cancer patients, according to geNorm and NormFinder respectively. Conclusion We propose a standardized approach of finding the most suitable reference gene(s) in every qRT-PCR experiment using TLDA.
Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)
Background Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation. Results Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable. Conclusion We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.
de Boer, Muriel E; de Boer, Tjalf E; Marien, Janine; Timmermans, Martijn JTN; Nota, Benjamin; van Straalen, Nico M; Ellers, Jacintha; Roelofs, Dick
Expression profiling of a high-fertility mouse line by microarray analysis and qPCR
Background In a recent study it was demonstrated that a largely increased ovulation number is responsible for high prolificacy in two mouse lines selected for fertility performance. The objective of the present study was to identify genes that are involved in increasing the ovulation number in one of these lines, FL1. For differential expression profiling, ovaries of FL1 and of a non-selected control line, DUKsi, both lines derived from the same genetic pool, were analyzed with microarray analysis and quantitative polymerase chain reaction (qPCR). Ovaries from 30 animals of each line were collected at the metestrous stage, combined to 6 pools each, and processed for microarray analysis. Results The actual number of ova shed in FL1 exceeded that of the DUKsi control line more than twofold (26.6 vs. 12.9). 148 differentially expressed ovarian transcripts could be identified, 74 of them up- and 74 down-regulated. Of these, 47 significantly mapped to specific Gene Ontology (GO) terms representing different biological processes as steroid metabolism, folliculogenesis, immune response, intracellular signal transduction (particularly of the G protein signaling cascade), regulation of transcription and translation, cell cycle and others. qPCR was used to re-evaluate selected transcripts and to estimate inter-individual variation of expression levels. These data significantly correlated with microarray data in 12 out of 15 selected transcripts but revealed partly large variations of expression levels between individuals. Conclusion (1) The abundance of numerous ovarian transcripts was significantly different in FL1 compared to the non-selected control line DUKsi thus suggesting that at least some of the respective genes and corresponding biological processes are involved in improving reproductive traits, particularly by increasing the number of ovulation. (2) Selective qPCR re-evaluation largely confirmed the microarray data and in addition demonstrated that sample pooling can be beneficial to find out group-specific expression profiles despite of large inter-individual variation. (3) The present data will substantially help ongoing genetic association studies to identify candidate genes and causative mutations responsible for increased fertility performance in mice.
Vanselow, Jens; Nurnberg, Gerd; Koczan, Dirk; Langhammer, Martina; Thiesen, Hans-Jurgen; Reinsch, Norbert
Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA
The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.
Fernandez-Jimenez, Nora; Castellanos-Rubio, Ainara; Plaza-Izurieta, Leticia; Gutierrez, Galder; Irastorza, Inaki; Castano, Luis; Vitoria, Juan Carlos; Bilbao, Jose Ramon
Reference Centiles for Serum Ferritin and Percentage of Transferrin Saturation, with Application to Mutations of the HFE Gene
Background: The gene that causes most cases of hered- itary hemochromatosis is designated HFE. Individuals with mutations in the HFE gene may have increased serum iron, transferrin saturation, and ferritin concen- trations relative to individuals with the wild-type geno- type. Methods: We generated reference centiles for percent- age of transferrin saturation and serum ferritin concen- trations in normal (wild-type), healthy
James A. Koziol; Ngoc J. Ho; Vincent J. Felitti; Ernest Beutler
Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions.
Real-time PCR is a highly sensitive, relatively easy to perform assay for quantifying mRNA abundance. However, there are several complexities built into the assay that can affect data interpretation. Most notably, the selection of an appropriate internal control for normalization is essential for expression data interpretation. In this study we investigated the suitability of seven commonly used genes [18S ribosomal RNA (18S), alpha tubulin (TUBA), beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), and RNA polymerase II polypeptide B (POLR2B)] as potential quantitative references for normalizing real-time PCR data generated in the study of channel catfish physiology. Gene expression and stability were evaluated among 15 channel catfish tissues and within physiologically-relevant tissues in response to experimental manipulation (i.e. LHRH injection, fasting, and acute stress). Expression of the seven candidate reference genes varied across all tissue types tested, indicating that none of the genes could suitably serve as reference genes for cross tissue comparisons. Experimentally altering the physiological state of the fish differentially affected expression of the various reference genes depending on experimental design and tissue type, with 18S unaffected by the experimental treatment in all tissues examined. For example, the selection of a differentially expressed gene, GAPDH, as opposed to 18S, to normalize hepatic growth hormone receptor during fasting resulted in misinterpretation of the data. These results reveal the importance of providing comprehensive details of reference gene validation when publishing real-time PCR results, with this manuscript serving as a basic guideline for reference gene selection in channel catfish research. PMID:18692590
Small, Brian C; Murdock, Christopher A; Bilodeau-Bourgeois, A Lelania; Peterson, Brian C; Waldbieser, Geoffrey C
Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes
Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep.
Reference Genes to Study Herbicide Stress Response in Lolium sp.: Up-Regulation of P450 Genes in Plants Resistant to Acetolactate-Synthase Inhibitors
Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants.
Duhoux, Arnaud; Delye, Christophe
Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection.
The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed. PMID:19902949
Paternň, Annalisa; Marchesi, Ugo; Gatto, Francesco; Verginelli, Daniela; Quarchioni, Cinzia; Fusco, Cristiana; Zepparoni, Alessia; Amaddeo, Demetrio; Ciabatti, Ilaria
Comparative Expression Profiling of the Chlamydia trachomatis pmp Gene Family for Clinical and Reference Strains
Background Chlamydia trachomatis, an obligate intracellular pathogen, is a leading worldwide cause of ocular and urogenital diseases. Advances have been made in our understanding of the nine-member polymorphic membrane protein (Pmp) gene (pmp) family of C. trachomatis. However, there is only limited information on their biologic role, especially for biological variants (biovar) and clinical strains. Methodology/Principal Findings We evaluated expression for pmps throughout development for reference strains E/Bour and L2/434, representing different biovars, and for clinical E and L2 strains. Immunoreactivity of patient sera to recombinant (r)Pmps was also determined. All pmps were expressed at two hours. pmpA had the lowest expression but was up-regulated at 12 h for all strains, indicating involvement in reticulate body development. For pmpD, expression peaked at 36 h. Additionally, 57.7% of sera from infected and 0% from uninfected adolescents were reactive to rPmpD (p?=?0.001), suggesting a role in immunogenicity. pmpF had the highest expression levels for all clinical strains and L2/434 with differential expression of the pmpFE operon for the same strains. Sera were nonreactive to rPmpF despite immunoreactivity to rMOMP and rPmpD, suggesting that PmpF is not associated with humoral immune responses. pmpFE sequences for clinical strains were identical to those of the respective reference strains. We identified the putative pmpFE promoter, which was, surprisingly, 100% conserved for all strains. Analyses of ribosomal binding sites, RNase E, and hairpin structures suggested complex regulatory mechanism(s) for this >6 Kb operon. Conclusions/Significance The dissimilar expression of the same pmp for different C. trachomatis strains may explain different strain-specific needs and phenotypic distinctions. This is further supported by the differential immunoreactivity to rPmpD and rPmpF of sera from patients infected with different strains. Furthermore, clinical E strains did not correlate with the E reference strain at the gene expression level, reinforcing the need for expansive studies of clinical strains.
Nunes, Alexandra; Gomes, Joao P.; Mead, Sally; Florindo, Carlos; Correia, Helena; Borrego, Maria J.; Dean, Deborah
Development of plasmid calibrators for absolute quantification of miRNAs by using real-time qPCR.
MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 25 nucleotides in length that negatively regulate gene expression via either the degradation or translational inhibition of their target mRNAs. Because miRNAs are essential for the regulation of critical physiological processes as well as a variety of pathological events, they have emerged as a novel class of molecular diagnostic biomarkers and therapeutic agents or targets. Accordingly, the need for novel methods for the quantification of miRNA has increased due to interest in their clinical implications. Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the most robust technology for nucleic acid quantification. Different tools for miRNA quantification by using qPCR are now commercially available, but only relative quantification strategies have been reported. This situation may be partly due to the difficulty in obtaining an appropriate molecule with which to establish an miRNA calibration range. Here, we describe a rapid and convenient strategy for the development of a calibrator, which enables the absolute quantification of miRNAs by using qPCR and allows the cloning of a synthetic sequence of interest instead of a PCR product into a plasmid. PMID:22642897
Formisano-Tréziny, Christine; de San Feliciano, Marina; Gabert, Jean
Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli.
Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/?l in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs. PMID:20674177
Peleg, Ofer; Baneth, Gad; Eyal, Osnat; Inbar, Jacob; Harrus, Shimon
Reference genes for reliable potyvirus quantitation in cassava and analysis of Cassava brown streak virus load in host varieties.
A reliable method for detection and quantitation of viruses associated with cassava brown streak disease (CBSD) is essential to determine their presence in material used for field propagation as well as for precise evaluation of CBSD resistance in the cassava germplasm. Quantitative RT-PCR (RT-qPCR) is a well-established method for precise quantitation of viral RNA amount in infected tissues. The method requires host reference genes with stable expression patterns under experimental conditions as internal controls for correct data normalization. Using the Genevestigator Refgene tool with Arabidopsis microarray data from Potyvirus-infected Arabidopsis as input data, candidate reference genes with stable expression pattern were selected as potential internal controls for the cassava -Cassava brown streak virus (CBSV; genus Ipomovirus; family Potyviridae) pathosystem. Primer pairs were designed for the cassava orthologs and their expression was analyzed in different tissues of three different CBSV-infected cassava varieties. The expression patterns of PP2A, UBQ10 and GTPb appeared to be the most stable in different CBSV-infected tissues and cassava varieties. The reference genes can therefore be used as internal controls for normalization of gene expression data in all types of cassava samples as well as in different cassava varieties infected by CBSV. The selected reference genes were used as internal controls to quantify CBSV in various symptomatic and asymptomatic plant organs to establish a correlation between virus load and symptom severity. PMID:21756941
Moreno, Isabel; Gruissem, Wilhelm; Vanderschuren, Hervé
Reference Gene Selection for Real-Time Quantitative PCR Analysis of the Mouse Uterus in the Peri-Implantation Period
The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNormPLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.
Lin, PengFei; Lan, XiangLi; Chen, FengLei; Yang, YanZhou; Jin, YaPing; Wang, AiHua
Expression Stabilities of Candidate Reference Genes for RT-qPCR under Different Stress Conditions in Soybean.
Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed. PMID:24124481
Ma, Shuhua; Niu, Hongwei; Liu, Chunji; Zhang, Jie; Hou, Chunyan; Wang, Dongmei
Expression Stabilities of Candidate Reference Genes for RT-qPCR under Different Stress Conditions in Soybean
Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed.
Liu, Chunji; Zhang, Jie; Hou, Chunyan; Wang, Dongmei
Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees.
A SYBR Green(®)-based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and ?-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR. PMID:23730001
Gadiou, S; Kundu, J K
Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l.) during temperature stress
Background One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment. Findings We here describe the expression of eight candidate reference genes from Atlantic cod (Gadus morhua l.) and their stability during thermal stress (temperature elevation of one degree C/day for 5 days). The genes investigated were: Eukaryotic elongation factor 1 alpha, ef1a; 18s ribosomal RNA; 18s, Ubiquitin conjugate protein; ubiq, cytoskeletal beta-actin; actb, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; b2m, cytoskeletal alpha-tubulin; tba1c, acidic ribosomal phosphoprotein; rplp1, glucose-6-phosphate dehydrogenase; g6pd. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills) from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: ubiq, ef1a, 18s and rplp1. We therefore used these to analyze the expression of stress related genes (heat shock proteins) induced during hyperthermia. We found that both transcripts were significantly upregulated in several tissues in fish exposed to increased temperature. Conclusion This is the first study comparing reference genes for RT-qPCR analyses of expression during hyperthermia in Atlantic cod. ef1a, 18s, rplp1 and ubiq transcripts were found to be well suited as reference genes during these experimental conditions.
Conceptual framework and pilot study to benchmark phylogenomic databases based on reference gene trees
Phylogenomic databases provide orthology predictions for species with fully sequenced genomes. Although the goal seems well-defined, the content of these databases differs greatly. Seven ortholog databases (Ensembl Compara, eggNOG, HOGENOM, InParanoid, OMA, OrthoDB, Panther) were compared on the basis of reference trees. For three well-conserved protein families, we observed a generally high specificity of orthology assignments for these databases. We show that differences in the completeness of predicted gene relationships and in the phylogenetic information are, for the great majority, not due to the methods used, but to differences in the underlying database concepts. According to our metrics, none of the databases provides a fully correct and comprehensive protein classification. Our results provide a framework for meaningful and systematic comparisons of phylogenomic databases. In the future, a sustainable set of 'Gold standard' phylogenetic trees could provide a robust method for phylogenomic databases to assess their current quality status, measure changes following new database releases and diagnose improvements subsequent to an upgrade of the analysis procedure.
Robinson-Rechavi, Marc; Xenarios, Ioannis; Dessimoz, Christophe
Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha
Background Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur. Results Eight genes were selected from a cDNA library of ovaries of B. brizantha considering the similarity to reference genes: EF1 (elongation factor 1 alpha), E1F4A (eukaryotic initiation factor 4A), GAPDH (glucose-6-phosphate dehydrogenase), GDP (glyceroldehyde-3-phosphate dehydrogenase), SUCOA (succinyl-CoA ligase), TUB (tubulin), UBCE (ubiquitin conjugating enzyme), UBI (ubiquitin). For the analysis, total RNA was extracted from 22 samples and raw Ct data after qRT-PCR reaction was analyzed for primer efficiency and for an overall analysis of Ct range among the different samples. Elongation factor 1 alpha showed the highest expression levels, whereas succinyl-CoA ligase showed the lowest within the chosen set of samples. GeNorm application was used for evaluation of the best reference genes, and according to that, the least stable genes, with the highest M values were tubulin and succinyl-CoA ligase and the most stable ones, with the lowest M values were elongation factor 1 alpha and ubiquitin conjugating enzyme, when both reproductive and vegetative samples were tested. For ovaries and spikelets of both sexual and apomictic B. brizantha the genes with the lowest M values were BbrizUBCE, BbrizE1F4A and BbrizEF1. Conclusion In total, eight genes belonging to different cellular processes were tested. Out of them, BbrizTUB was the less stable while BbrizEF1 followed by BbrizUBCE were the more stable genes considering male and female reproductive tissues, spikelets, roots and leaves. Regarding the best reference genes for ovary tissues, where apomictic and sexual reproduction must occur, the best reference genes were BbrizUBCE, BbrizE1F4A and BbrizEF1. Our results provide crucial information for transcriptional analysis in the Brachiaria ssp, helping to improve the quality of gene expression data in these species, which constitute an excellent plant system for the study of apomixis.
Silveira, Erica Duarte; Alves-Ferreira, Marcio; Guimaraes, Larissa Arrais; da Silva, Felipe Rodrigues; Carneiro, Vera Tavares de Campos
Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis
The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For\\u000a this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically\\u000a validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic\\u000a pterostilbene, which plays a crucial role in
Magdalena Gamm; Marie-Claire Héloir; Jani Kelloniemi; Benoît Poinssot; David Wendehenne; Marielle Adrian
Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants.
The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants. PMID:23552195
Bonnet, M; Bernard, L; Bes, S; Leroux, C
The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.
Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown. PMID:18433420
Gutierrez, Laurent; Mauriat, Mélanie; Guénin, Stéphanie; Pelloux, Jérôme; Lefebvre, Jean-François; Louvet, Romain; Rusterucci, Christine; Moritz, Thomas; Guerineau, François; Bellini, Catherine; Van Wuytswinkel, Olivier
Seasonal Dynamics of Microcystis spp. and Their Toxigenicity as Assessed by qPCR in a Temperate Reservoir
Blooms of toxic cyanobacteria are becoming increasingly frequent, mainly due to water quality degradation. This work applied qPCR as a tool for early warning of microcystin(MC)-producer cyanobacteria and risk assessment of water supplies. Specific marker genes for cyanobacteria, Microcystis and MC-producing Microcystis, were quantified to determine the genotypic composition of the natural Microcystis population. Correlations between limnological parameters, pH, water temperature, dissolved oxygen and conductivity and MC concentrations as well as Microcystis abundance were assessed. A negative significant correlation was observed between toxic (with mcy genes) to non-toxic (without mcy genes) genotypes ratio and the overall Microcystis density. The highest proportions of toxic Microcystis genotypes were found 4–6 weeks before and 8–10 weeks after the peak of the bloom, with the lowest being observed at its peak. These results suggest positive selection of non-toxic genotypes under favorable environmental growth conditions. Significant positive correlations could be found between quantity of toxic genotypes and MC concentration, suggesting that the method applied can be useful to predict potential MC toxicity risk. No significant correlation was found between the limnological parameters measured and MC concentrations or toxic genotypes proportions indicating that other abiotic and biotic factors should be governing MC production and toxic genotypes dynamics. The qPCR method here applied is useful to rapidly estimate the potential toxicity of environmental samples and so, it may contribute to the more efficient management of water use in eutrophic systems.
Martins, Antonio; Moreira, Cristiana; Vale, Micaela; Freitas, Marisa; Regueiras, Ana; Antunes, Agostinho; Vasconcelos, Vitor
Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays
Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels. One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay. Automated qPCR setup has improved this field by allowing for greater reproducibility. Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment. This method along with internal plate controls also reduces experimental variables common to other techniques. We recently developed a qPCR assay for profiling of pre-microRNAs (pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level. These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful. There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique. Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing. Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.
Dittmer, Dirk P
Stable Internal Reference Genes for the Normalization of Real-Time PCR in Different Sweetpotato Cultivars Subjected to Abiotic Stress Conditions
Reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most widely used methods for gene expression analysis, but its successful application depends on the stability of suitable reference genes used for data normalization. In plant studies, the choice and optimal number of reference genes must be experimentally determined for the specific conditions, plant species, and cultivars. In this study, ten candidate reference genes of sweetpotato (Ipomoea batatas) were isolated and the stability of their expression was analyzed using two algorithms, geNorm and NormFinder. The samples consisted of tissues from four sweetpotato cultivars subjected to four different environmental stress treatments, i.e., cold, drought, salt and oxidative stress. The results showed that, for sweetpotato, individual reference genes or combinations thereof should be selected for use in data normalization depending on the experimental conditions and the particular cultivar. In general, the genes ARF, UBI, COX, GAP and RPL were validated as the most suitable reference gene set for every cultivar across total tested samples. Interestingly, the genes ACT and TUB, although widely used, were not the most suitable reference genes in different sweetpotato sample sets. Taken together, these results provide guidelines for reference gene(s) selection under different experimental conditions. In addition, they serve as a foundation for the more accurate and widespread use of RT-qPCR in various sweetpotato cultivars.
Ji, Chang Yoon; Park, Seyeon; Jeong, Jae cheol; Lee, Haeng-Soon; Kwak, Sang-Soo
Reference gene selection for real-time RT-PCR in regenerating mouse livers
The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.
Tatsumi, Kohei [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan); Ohashi, Kazuo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)], E-mail: email@example.com; Taminishi, Sanae [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan); Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Yoshioka, Akira; Shima, Midori [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan)
Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)
BACKGROUND: Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate
Julia M Lee; John R Roche; Danny J Donaghy; Anthony Thrush; Puthigae Sathish
Identification of stable reference genes for gene expression studies using quantitative real time PCR in buffalo oocytes and embryos.
The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15,RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes?embryos. The information would help in more accurate interpretation of gene expression data from oocytes?embryos towards understanding the molecular events in these cells during development. PMID:23289123
Kumar, Parveen; Yadav, Poonam; Verma, Arpana; Singh, Dheer; De, Sachinandan; Datta, Tirtha Kumar
Selection of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Cartilage Tissue Injury and Repair in Rabbits
When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and ?-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues.
Peng, Xiao-Xiang; Zhao, Rong-Lan; Song, Wei; Chu, Hai-Rong; Li, Meng; Song, Shu-Ya; Li, Guang-Zhou; Liang, Dong-Chun
Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers
Background Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant\\u000a virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral\\u000a transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative\\u000a gene expression analysis. In order to normalize
Guillermo A Maroniche; Mónica Sagadín; Vanesa C Mongelli; Graciela A Truol; Mariana del Vas
Application of absolute qPCR as a screening method to detect illicit 17?-oestradiol administration in male cattle.
It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17?-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17?-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CC?, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n = 12), consisting of animals treated with four doses of 17?-oestradiol (5 mg week(-1) per animal); and group B (n = 20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17?-oestradiol (group C; 20 mg week(-1) per animal; n = 6) or remained untreated (group D; n = 20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CC? on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CC? and they were classified as being suspected of 17?-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium. PMID:23131142
Uslenghi, F; Divari, S; Cannizzo, F T; De Maria, R; Spada, F; Mulasso, C; Pezzolato, M; Bozzetta, E; Attucci, A; Giorgi, P; Biolatti, B
Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples
BACKGROUND: Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. METHODS: We performed single-end RNA-Seq on three human prostate
Serban Nacu; Wenlin Yuan; Zhengyan Kan; Deepali Bhatt; Celina Rivers; Jeremy Stinson; Brock A Peters; Zora Modrusan; Kenneth Jung; Somasekar Seshagiri; Thomas D Wu
MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples
DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from ...
Comparison of culture and qPCR methods in detection of mycobacteria from drinking waters.
Environmental mycobacteria are common bacteria in man-made water systems and may cause infections and hypersensitivity pneumonitis via exposure to water. We compared a generally used cultivation method and a quantitative polymerase chain reaction (qPCR) method to detect mycobacteria in 3 types of drinking waters: surface water, ozone-treated surface water, and groundwater. There was a correlation between the numbers of mycobacteria obtained by cultivation and qPCR methods, but the ratio of the counts obtained by the 2 methods varied among the types of water. The qPCR counts in the drinking waters produced from surface or groundwater were 5 to 34 times higher than culturable counts. In ozone-treated surface waters, both methods gave similar counts. The ozone-treated drinking waters had the highest concentration of assimilable organic carbon, which may explain the good culturability. In warm tap waters, qPCR gave 43 times higher counts than cultivation, but both qPCR counts and culturable counts were lower than those in the drinking waters collected from the same sites. The TaqMan qPCR method is a rapid and sensitive tool for total quantitation of mycobacteria in different types of clean waters. The raw water source and treatments affect both culturability and total numbers of mycobacteria in drinking waters. PMID:23586753
Räsänen, Noora H J; Rintala, Helena; Miettinen, Ilkka T; Torvinen, Eila
Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q).
The ability to reliably analyze cellular and molecular profiles of normal or diseased tissues is frequently complicated by the inherent heterogeneous nature of tissues. Laser Capture Microdissection (LCM) is an innovative technique that allows the isolation and enrichment of pure subpopulations of cells from tissues under direct microscopic examination. Material obtained by LCM can be used for downstream assays including gene microarrays, western blotting, cDNA library generation and DNA genotyping. We describe a series of LCM protocols for cell collection, RNA extraction and qPCR gene expression analysis. Using reagents we helped develop commercially, we focus on two LCM approaches: laser cutting and laser capture. Reagent calculations have been pre-determined for 10 samples using the new PREXCEL-Q assay development and project management software. One can expect the entire procedure for laser cutting coupled to qPCR to take approximately 12.5-15 h, and laser capture coupled to qPCR to take approximately 13.5-17.5 h. PMID:20556230
Sow, Fatoumata B; Gallup, Jack M; Sacco, Randy E; Ackermann, Mark R
RefNetBuilder: a platform for construction of integrated reference gene regulatory networks from expressed sequence tags
Background Gene Regulatory Networks (GRNs) provide integrated views of gene interactions that control biological processes. Many public databases contain biological interactions extracted from experimentally validated literature reports, but most furnish only information for a few genetic model organisms. In order to provide a bioinformatic tool for researchers who work with non-model organisms, we developed RefNetBuilder, a new platform that allows construction of putative reference pathways or GRNs from expressed sequence tags (ESTs). Results RefNetBuilder was designed to have the flexibility to extract and archive pathway or GRN information from public databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG). It features sequence alignment tools such as BLAST to allow mapping ESTs to pathways and GRNs in model organisms. A scoring algorithm was incorporated to rank and select the best match for each query EST. We validated RefNetBuilder using DNA sequences of Caenorhabditis elegans, a model organism having manually curated KEGG pathways. Using the earthworm Eisenia fetida as an example, we demonstrated the functionalities and features of RefNetBuilder. Conclusions The RefNetBuilder provides a standalone application for building reference GRNs for non-model organisms on a number of operating system platforms with standard desktop computer hardware. As a new bioinformatic tool aimed for constructing putative GRNs for non-model organisms that have only ESTs available, RefNetBuilder is especially useful to explore pathway- or network-related information in these organisms.
Injury Modality, Survival Interval, and Sample Region Are Critical Determinants of qRT-PCR Reference Gene Selection during Long-Term Recovery from Brain Trauma
Abstract In the present study we examined expression of four real-time quantitative RT-PCR reference genes commonly applied to rodent models of brain injury. Transcripts for ?-actin, cyclophilin A, GAPDH, and 18S rRNA were assessed at 2–15 days post-injury, focusing on the period of synaptic recovery. Diffuse moderate central fluid percussion injury (FPI) was contrasted with unilateral entorhinal cortex lesion (UEC), a model of targeted deafferentation. Expression in UEC hippocampus, as well as in FPI hippocampus and parietotemporal cortex was analyzed by qRT-PCR. Within-group variability of gene expression was assessed and change in expression relative to paired controls was determined. None of the four common reference genes tested was invariant across brain region, survival time, and type of injury. Cyclophilin A appeared appropriate as a reference gene in UEC hippocampus, while ?-actin was most stable for the hippocampus subjected to FPI. However, each gene may fail as a suitable reference with certain test genes whose RNA expression is targeted for measurement. In FPI cortex, all reference genes were significantly altered over time, compromising their utility for time-course studies. Despite such temporal variability, certain genes may be appropriate references if limited to single survival times. These data provide an extended baseline for identification of appropriate reference genes in rodent studies of recovery from brain injury. In this context, we outline additional considerations for selecting a qRT-PCR normalization strategy in such studies. As previously concluded for acute post-injury intervals, we stress the importance of reference gene validation for each brain injury paradigm and each set of experimental conditions.
Harris, Janna L.; Reeves, Thomas M.
Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search
Background The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated. We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system – specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. Results We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. Conclusions In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.
A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification
Background Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.
Rutledge, Robert G.
Evaluation of Reference Genes for Expression Studies in Ash ( Fraxinus spp.)
Ash (Fraxinus spp.) is a dominant tree species in North America, in both managed and natural landscapes. However, due to the rapid invasion\\u000a by the emerald ash borer (Agrilus planipennis), an exotic invasive insect pest, millions of North American ash trees have been killed. Real-time quantitative polymerase\\u000a chain reaction (RTq-PCR) is widely used for validating transcript levels in gene expression
Loren Rivera-Vega; Praveen Mamidala; Jennifer L. Koch; Mary E. Mason; Omprakash Mittapalli
Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-? mRNA expression.
Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-?, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions. PMID:21510983
Martino, Alessandro; Cabiati, Manuela; Campan, Manuela; Prescimone, Tommaso; Minocci, Daiana; Caselli, Chiara; Rossi, Anna Maria; Giannessi, Daniela; Del Ry, Silvia
Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say)
Background L. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato. Results The expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADP-ribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1? EF1?) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR. Conclusions The expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata.
Differentiating microbial forensic qPCR target and control products by electrospray ionization mass spectrometry.
Molecular bioforensic research is dependent on rapid and sensitive methods such as real-time PCR (qPCR) for the identification of microorganisms. The use of synthetic positive control templates containing small modifications outside the primer and probe regions is essential to ensure all aspects of the assay are functioning properly, including the primers and probes. However, a typical qPCR or reverse transcriptase qPCR (qRT-PCR) assay is limited in differentiating products generated from positive controls and biological samples because the fluorescent probe signals generated from each type of amplicon are indistinguishable. Additional methods used to differentiate amplicons, including melt curves, secondary probes, and amplicon sequencing, require significant time to implement and validate and present technical challenges that limit their use for microbial forensic applications. To solve this problem, we have developed a novel application of electrospray ionization mass spectrometry (ESI-MS) to rapidly differentiate qPCR amplicons generated with positive biological samples from those generated with synthetic positive controls. The method has sensitivity equivalent to qPCR and supports the confident and timely determination of the presence of a biothreat agent that is crucial for policymakers and law enforcement. Additionally, it eliminates the need for time-consuming methods to confirm qPCR results, including development and validation of secondary probes or sequencing of small amplicons. In this study, we demonstrate the effectiveness of this approach with microbial forensic qPCR assays targeting multiple biodefense agents (bacterial, viral, and toxin) for the ability to rapidly discriminate between a positive control and a positive sample. PMID:23675878
Motley, S Timothy; Redden, Cassie L; Sannes-Lowery, Kristin A; Eshoo, Mark W; Hofstadler, Steven A; Burans, James P; Rosovitz, M J
Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR
Using ammonia monooxygenase ?-subunit (amoA) gene and 16S rRNA gene, the community structure and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing\\u000a bacteria (AOB) in a nitrogen-removing reactor, which was operated for five phases, were characterized and quantified by cloning,\\u000a terminal restriction fragment length polymorphism (T-RFLP), and quantitative polymerase chain reaction (qPCR). The results\\u000a suggested that the dominant AOB in the
Tao Jin; Tong Zhang; Qingmei Yan
Selection of internal reference genes for normalization of quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in the canine brain and other organs.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive technique for quantifying gene expression. To analyze qRT-PCR data accurately, suitable reference genes that show consistent expression patterns across different tissues and experimental conditions should be selected. The objective of this study was to obtain the most stable reference genes in dogs, using samples from 13 different brain tissues and 10 other organs. 16 well-known candidate reference genes were analyzed by the geNorm, NormFinder, and BestKeeper programs. Brain tissues were derived from several different anatomical regions, including the forebrain, cerebrum, diencephalon, hindbrain, and metencephalon, and grouped accordingly. Combination of the three different analyses clearly indicated that the ideal reference genes are ribosomal protien S5 (RPS5) in whole brain, RPL8 and RPS5 in whole body tissues, RPS5 and RPS19 in the forebrain and cerebrum, RPL32 and RPS19 in the diencephalon, GAPDH and RPS19 in the hindbrain, and MRPS7 and RPL13A in the metencephalon. These genes were identified as ideal for the normalization of qRT-PCR results in the respective tissues. These findings indicate more suitable and stable reference genes for future studies of canine gene expression. PMID:22531949
Park, Sang-Je; Huh, Jae-Won; Kim, Young-Hyun; Lee, Sang-Rae; Kim, Sang-Hyun; Kim, Sun-Uk; Kim, Heui-Soo; Kim, Min Kyu; Chang, Kyu-Tae
Reference genes evaluated for use in infectious pancreatic necrosis virus real-time RT-qPCR assay applied during different stages of an infection
The stability of 6 reference genes, 18S, ?-actin, RPS20, eEF1?, G6PDH and GAPDH, was examined in tissues from Atlantic salmon (Salmo salar) and Chinook salmon embryo cells (CHSE-214). The main objective of this study was to determine the most suitable reference genes for use for the normalisation of data in quantitative real-time RT-qPCR assays conducted on infected tissues. The tissue
K. Julin; L.-H. Johansen; A.-I. Sommer
Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears
The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.
Tasker, Severine; Peters, Iain R.; Mumford, Andrew D.; Day, Michael J.; Gruffydd-Jones, Timothy J.; Day, Sarinder; Pretorius, Anne-Marie; Birtles, Richard J.; Helps, Chris R.; Neimark, Harold
Addressing fluorogenic real-time qpcr inhibition using the novel custom excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition\\u000a and to introduce\\/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and\\u000a enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems\\u000a this invention solves are universal to all qPCR reactions, and
Jack M. Gallup; Mark R. Ackermann
Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.
The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs. PMID:20849891
Nylund, Lotta; Heilig, Hans G H J; Salminen, Seppo; de Vos, Willem M; Satokari, Reetta
Validation of hisH4 and cox5 reference genes for RT-qPCR analysis of gene expression in Aspergillus flavus under aflatoxin conducive and non-conducive conditions.
Aspergillus flavus is an environmental pathogen that produces highly carcinogenic aflatoxins. Biosynthesis of aflatoxins is affected by external factors such as pH, temperature, carbon source and nitrogen source. Real-Time PCR (RT-qPCR) is a powerful technique used to detect minute changes in gene expression of a target gene in comparison to one or more reference genes. Several candidate genes were analysed to determine their suitability for use as reference genes for analysing gene expression in A. flavus via RT-qPCR under various aflatoxin conducive and non-conducive conditions. BestKeeper analysis indicated that histone H4 (hisH4) and cytochrome C oxidase subunit V (cox5) were suitable reference genes for analysis of gene expression in A. flavus via RT-qPCR. This was further confirmed by REST2009 analysis of hisH4 and cox5 stability. Furthermore, REST2009 was used to predict which gene or gene combination would be the best reference gene/s for RT-qPCR expression analysis under each treatment condition tested in this study. PMID:22704686
Suleman, Essa; Somai, Benesh Munilal
Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions
Background Reverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species. Results Relative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene. Conclusion Although the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.
Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids
BACKGROUND: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression
Lies Bogaert; Mario Van Poucke; Cindy De Baere; Luc Peelman; Frank Gasthuys; Ann Martens
Structural gene analysis of a Quebec reference strain or porcine reproductive and respiratory syndrome virus (PRRSV).
The 3' end genomic region of a Québec PRRSV reference strain (IAF-exp91), propagated in porcine alveolar macrophages (PAM), was sequenced and compared to the prototype European strain, the Lelystad virus (LV). The sequence, which represents the 3'-terminal 2834 nucleotides, encompassed 5 ORFs corresponding to ORFs 3 to 7 of LV. Extensive genomic variations resulting from an important rate of nucleotide additions, substitutions, and deletions were demonstrated between the two viruses. Indeed, the two corresponding sequences displayed a total of 66% and 63% identity at the nucleotide and amino acid levels, respectively. The predicted products of ORFs 5, 3, and 7, showed the highest rate of amino acid variations with percentages of identity of 52, 54, and 59, respectively. Sequence analysis of an additional Québec strain that could be propagated in a continuous cell line (MARC-145), suggested that Québec PRRSV strains belong to a genotype distinct from that of LV, thus confirming previous serological results which allowed to divide PRRSV isolates into two distinct antigenic subgroups (U.S. and European). Six viral major polypeptides with apparent M(rs) of 14.5K, 15K, 19K, 24.5K, 29K, and 42K could be identified from lysates of viral infected cells, of which the 15, 19 and 24.5K species seemed to be structural. In vitro translation products of ORFs 7 and 6 comigrated with the 15 and 19 K viral proteins, whereas that of ORF 5 may be associated to the 24.5K when translated in presence of microsomes. Consequently, it is likely that ORFs 7 to 5, encode the three major structural proteins. PMID:8830492
Mardassi, H; Mounir, S; Dea, S
The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number
The quantitative polymerase chain reaction (QPCR) assay allows measurement of DNA damage in the mitochondrial and nuclear genomes without isolation of mitochondria. It also permits measurement of relative mitochondrial genome copy number. Finally, it can be used for measurement of DNA repair in vivo when employed appropriately. In this manuscript we briefly review the methodology of the QPCR assay, discuss its strengths and limitations, address considerations for measurement of mitochondrial DNA repair, and describe methodological changes implemented in recent years. We present QPCR assay primers and reaction conditions for five species not previously described in a Methods article: Caenorhabditis elegans, Fundulus heteroclitus, Danio rerio, Drosophila melanogaster, and adenovirus. Finally, we illustrate the use of the assay by measuring repair of ultraviolet C radiation-induced DNA damage in the nuclear but not mitochondrial genomes of a zebrafish cell culture.
Hunter, Senyene E.; Jung, Dawoon; Di Giulio, Richard T.; Meyer, Joel N.
Selection of optimal reference genes for expression analysis in the entomopathogenic fungus Beauveria bassiana during development, under changing nutrient conditions, and after exposure to abiotic stresses.
The selection of suitable reference genes is crucial for accurate quantification of gene expression. To identify suitable reference genes in Beauveria bassiana, the expression of 14 candidates (18S, 28S, ?-Tub, GAPD, ?-Act, TEF, HGPT, His3, His2A, TBP, CypA, CypB, PP1, and CrzA) was measured by quantitative polymerase chain reaction at different development stages and under various nutritional and stress conditions. Expression stability, as evaluated by the geNorm and NormFinder programs, revealed that His2A/?-Act/CrzA was the most stably expressed set of genes throughout development, while 28S/PP1/CypA and His2A/?-Act/CypA were the most stably expressed gene sets under a variety of nutritional and stress conditions, respectively. Overall, the most stably expressed genes under all conditions examined were PP1, ?-Act, and CypA. PMID:21922424
Zhou, Yong-Hong; Zhang, Yong-Jun; Luo, Zhi-Bing; Fan, Yan-Hua; Tang, Gui-Rong; Liu, Li-Ji; Pei, Yan
Multiplex qPCR for Detection and Absolute Quantification of Malaria.
We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria. PMID:24009663
Kamau, Edwin; Alemayehu, Saba; Feghali, Karla C; Saunders, David; Ockenhouse, Christian F
Detection of a bacteriophage gene encoding a Mu-like portal protein in Haemophilus parasuis reference strains and field isolates by nested polymerase chain reaction.
A nested polymerase chain reaction (nPCR) assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene's sequence, were utilized. A majority of the virulent reference strains and field isolates tested harbored the gene. The results suggest that the nPCR technique described in the current report could serve as a tool for epidemiological studies of H. parasuis. PMID:21908286
Zehr, Emilie S; Tabatabai, Louisa B
Quantification of siRNA using competitive qPCR
We have developed a PCR-based short interfering RNA (siRNA) quantification method based on com- petition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could
Wei-li Liu; Mark Stevenson; Leonard W. Seymour; Kerry D. Fisher
Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development
Background Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. Methodology/Principal Findings We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (?-actin, cyclophilin b, ?-tubulin and lamin A/C), while MAPK1 was stably expressed. Conclusion Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.
Rocha-Martins, Mauricio; Njaine, Brian; Silveira, Mariana S.
Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional.
Ackermann, Mark R.
An improved qPCR protocol for rapid detection and quantification of Clostridium difficile in cattle feces.
Clostridium difficile (CD) can cause a significant and transmissible disease in animals and humans, with poorly understood epidemiology. Animals have been suggested as a possible source of infection and environment contamination. It is necessary that a precise and rapid diagnostic tool is available for the detection of CD from clinical and/or environmental samples. A quantitative real-time PCR (qPCR) protocol for CD detection defined by Penders et al. (FEMS Microbiol Lett, 243, 2005, 141-147) was modified. The modified protocol, supported by a novel extraction method, was tested on CD-spiked cattle feces and clinical fecal samples from calves. Quantification was performed targeting CD 16S rRNA gene. Three different commonly used TaqMan universal PCR master mixes were also compared. Results indicate that the modified protocol is very sensitive with an LOD of 7.72 CD cells per g CD-spiked feces. The protocol is capable of precise quantification with an LOQ of 77.2 CD cells per g CD-spiked feces, R(2) between 0.9957 and 0.9968, isolation efficiency from 87.89% to 90.96%, and an interassay CV ranging from 3.71% to 9.57%. The qPCR protocol for the detection and quantification of CD from animal feces investigated and described in this article using MIQE guidelines has the lowest detection and quantification limits published to date. Therefore, it can be implemented for precise epidemiological investigations of CD infections in animals and humans. PMID:23397963
Bandelj, Petra; Logar, Katarina; Usenik, Alenka M; Vengust, Modest; Ocepek, Matjaz
Validation of Reference Genes in Cervical Cell Samples from Human Papillomavirus-Infected and -Uninfected Women for Quantitative Reverse Transcription-PCR Assays?
Reference genes for quantitative reverse transcription-PCR (qRT-PCR) studies must be validated for the cell type studied and should be stable between the groups that represent the independent variable in an experimental design. We sought to identify the reference genes in cervical cell specimens showing the most stable expression between human papillomavirus (HPV)-infected and -uninfected women without high-grade cervical intraepithelial neoplasia. Using endocervical cells collected by cytology brush and Sybr green-based qRT-PCR, eight candidate genes were screened for amplification efficiency, specificity, and overall stability (by use of geNorm software). The five most stable genes were then further evaluated both for overall stability (geNorm) and intergroup stability (by use of NormFinder software) in specimens from HPV-negative and HPV-positive women. The combination of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and RPLP0 was the most stable overall, with a geNorm stability measure of 0.603. The intergroup analysis showed GAPDH to be the most stable single gene and RPLP0 to be second most stable and also showed that these genes represent the most stable two-gene combination, with a NormFinder stability value of 0.130. The fact that these two distinct approaches identified the same pair of genes provides added confidence that, when the focus is on HPV infection, a normalization factor derived from these two genes is likely to be appropriate.
Daud, Ibrahim I.; Scott, Mark E.
Quantitative PCR (QPCR) analysis of molds in indoor environmental samples produces highly accurate speciation and enumeration data. In a number of studies, eighty of the most common or potentially problematic indoor molds were identified and quantified in dust samples from homes...
Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR
Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide
Shawn T. Clark; Kimberley A. Gilbride; Mehrab Mehrvar; Andrew E. Laursen; Vadim Bostan; Ronald Pushchak; Lynda H. McCarthy
Quantification of Salmonella spp. in composted biosolids using a TaqMan qPCR assay
Composting is increasingly used to transform biosolids, obtained following wastewater treatment, into a more stable organic product that can be released in the environment. The process must however be closely monitored to assure that the end product meets the regulations set by environmental agencies with regards to the amount of pathogenic microorganisms present. In this study, a TaqMan qPCR approach
Amy Novinscak; Céline Surette; Martin Filion
MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples
DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any
Mano Sivaganesan; Shawn Siefring; Manju Varma; Richard A. Haugland
QPCR Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches
The use of real-time qPCR to determine fecal indicator bacteria (FIB) densities is currently being investigated by the U.S. EPA. The present recreational water quality guidelines, based on culturable FIB, prevent same day determinations of water quality whereas results from the ...
International collaborative study of the endogenous reference gene, sucrose phosphate synthase (SPS), used for qualitative and quantitative analysis of genetically modified rice.
One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates. PMID:19326953
Jiang, Lingxi; Yang, Litao; Zhang, Haibo; Guo, Jinchao; Mazzara, Marco; Van den Eede, Guy; Zhang, Dabing
Genetics Home Reference: Arrhythmia
... Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Arrhythmia Related topics on Genetics Home Reference: Andersen-Tawil ... You may also search Genetics Home Reference for "Arrhythmia" for additional information. Published: September 23, 2013 Lister ...
Quantification of siRNA using competitive qPCR
We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could not act as primer for amplicon generation, the lowest competing primer concentration yielding a linear relationship between template DNA amount (0.1–50 ng) and cycle of threshold (Ct) was determined (6.25 nM). Under these conditions addition of sequence-specific siRNA to the competitive quantitative PCR (cqPCR), resulted in a dose-dependent linear increase in Ct value. 2?-O-methyl ribose-modified siRNA retained an ability to inhibit template amplification in serum, unlike unmodified siRNAs that were susceptible to endonucleases. Mismatch-bearing or truncated siRNAs failed to inhibit template amplification confirming sequence specificity and an ability to discriminate between degraded and non-degraded siRNA sequences. Following delivery of E6 siRNA to C33-A cells using oligofectamine or His6 reducible polymers, siRNA uptake was quantified by cqPCR, revealing dose-dependent uptake. We anticipate that cqPCR will allow accurate determination of siRNA pharmacokinetics following in vivo delivery, greatly facilitating development of therapeutic siRNA delivery strategies.
Liu, Wei-li; Stevenson, Mark; Seymour, Leonard W.; Fisher, Kerry D.
Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples
During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.
Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.
Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne's disease.
A quantitative real-time PCR (qPCR) assay employing IS900 gene specific primers of Mycobacterium avium subsp. parartuberculosis (MAP) was compared with conventional PCR, bacterial culture and enzyme-linked immunosorbent assay in 38 sheep showing granulomatous enteritis and lymphadenitis with and without demonstration of acid-fast bacilli (AFB). The lesions were classified as multibacillary (MB) (n?=?23), which had diffuse granulomatous lesions with abundant AFB, and paucibacillary (PB) (n?=?15), which had focal or multifocal granulomatous lesions with few or no AFB. In the multibacillary group (MB), IS900 PCR detected 19 (82.6%), and qPCR detected all 23 (100%) sheep positive for MAP in the intestine and lymph node tissues. In the paucibacillary group (PB), IS900 PCR detected 2 (13.3%), and qPCR detected all 15 (100%) sheep positive for MAP in tissues. When results of both groups were taken together, IS900 PCR detected 21(55.2%), and qPCR detected all 38 (100%) animals positive for MAP genome either in the intestine or lymph node tissues. On Herrold egg yolk medium, tissues of 14 (60.9%) MB and 5 (33.3%) PB sheep were found to be positive for MAP. Out of 27 sheep (PB?=?8, MB?=?19) tested by an ELISA, 21 (77.7%) were found to be positive for MAP antibody, of which 25% (2/8) and 100% (19/19) sheep were from PB and MB sheep, respectively. Based on the results of the present study, it was concluded that qPCR was a highly sensitive test in comparison to conventional PCR, ELISA and bacterial culture for the diagnosis of paratuberculosis on infected tissues especially from paucibacillary sheep. PMID:23539663
Sonawane, Ganesh G; Tripathi, Bhupendra N
A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: A diagnostic tool for STR typing
A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a ?170–190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY),
William R. Hudlow; Mavis Date Chong; Katie L. Swango; Mark D. Timken; Martin R. Buoncristiani
Validation of an rpoB Gene PCR Assay for Detection of Tropheryma whipplei: 10 Years' Experience in a National Reference Laboratory.
The performance of a real-time PCR assay targeting the Tropheryma whipplei rpoB gene was evaluated using test strains and 1,236 clinical specimens in a national reference laboratory. The novel rpoB-PCR assay proved to be specific, revealed improved analytical sensitivity, and substantially accelerated detection of T. whipplei DNA in clinical specimens. PMID:23966507
Moter, Annette; Schmiedel, Dinah; Petrich, Annett; Wiessner, Alexandra; Kikhney, Judith; Schneider, Thomas; Moos, Verena; Göbel, Ulf B; Reischl, Udo
The expression of genes PKM2 and CAST in the muscle tissue of pigs differentiated by glycolytic potential and drip loss, with reference to the genetic group
The present studies aimed at an analysis of the expression level of genes PKM2 and CAST in Longissimus lumborum [LL] muscle tissue of pigs differing as regards the glycolytic potential [GP] and drip loss [DL] from the LL muscle, with reference to the genetic group. The studies covered a total of 65 pigs: 20 purebred Landrace [L], 22 crossbreeds of
H. Sieczkowska; A. Zybert; E. Krz?cio; K. Antosik; M. Ko?win-Podsiad?a; M. Pierzcha?a; P. Urba?ski
Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications.
RNA transcripts such as mRNA or microRNA are frequently used as biomarkers to determine disease state or response to therapy. Reverse transcription (RT) in combination with quantitative PCR (qPCR) has become the method of choice to quantify small amounts of such RNA molecules. In parallel with the democratization of RT-qPCR and its increasing use in biomedical research or biomarker discovery, we witnessed a growth in the number of gene expression data analysis methods. Most of these methods are based on the principle that the position of the amplification curve with respect to the cycle-axis is a measure for the initial target quantity: the later the curve, the lower the target quantity. However, most methods differ in the mathematical algorithms used to determine this position, as well as in the way the efficiency of the PCR reaction (the fold increase of product per cycle) is determined and applied in the calculations. Moreover, there is dispute about whether the PCR efficiency is constant or continuously decreasing. Together this has lead to the development of different methods to analyze amplification curves. In published comparisons of these methods, available algorithms were typically applied in a restricted or outdated way, which does not do them justice. Therefore, we aimed at development of a framework for robust and unbiased assessment of curve analysis performance whereby various publicly available curve analysis methods were thoroughly compared using a previously published large clinical data set (Vermeulen et al., 2009) [11]. The original developers of these methods applied their algorithms and are co-author on this study. We assessed the curve analysis methods' impact on transcriptional biomarker identification in terms of expression level, statistical significance, and patient-classification accuracy. The concentration series per gene, together with data sets from unpublished technical performance experiments, were analyzed in order to assess the algorithms' precision, bias, and resolution. While large differences exist between methods when considering the technical performance experiments, most methods perform relatively well on the biomarker data. The data and the analysis results per method are made available to serve as benchmark for further development and evaluation of qPCR curve analysis methods (http://qPCRDataMethods.hfrc.nl). PMID:22975077
Ruijter, Jan M; Pfaffl, Michael W; Zhao, Sheng; Spiess, Andrej N; Boggy, Gregory; Blom, Jochen; Rutledge, Robert G; Sisti, Davide; Lievens, Antoon; De Preter, Katleen; Derveaux, Stefaan; Hellemans, Jan; Vandesompele, Jo
Expression analysis of efflux pump genes among drug-susceptible and multidrug-resistant Mycobacterium tuberculosis clinical isolates and reference strains.
Finding a gene or genes that are involved with multidrug resistance will be useful for finding a new target for the treatment of drug resistant tuberculosis. In this study, we aimed to compare the differences of the expression of 15 putative multidrug efflux pump genes in clinically isolated drug sensitive and multidrug resistant (MDR) Mycobacterium tuberculosis isolates, and reference strains. We found that these genes in the drug-sensitive and MDR M. tuberculosis isolates have similar rates of expressions. However, we found the expression levels of the all the genes are significantly higher in the clinical strains compared to the expression level of genes in the reference strains. In addition to this, it is found that standard strain has lower MIC value for the drugs including streptomycin and rifampin compared to the clinical isolate. We presume that the increase of the gene expression in the clinical strains is due to the exposure of antituberculosis drugs during treatment of patients, which cause constitutive expression of efflux systems, which might increase MIC levels of the major anti-tuberculosis drugs. PMID:23561272
Calgin, Mustafa Kerem; Sahin, Fikret; Turegun, Buse; Gerceker, Devran; Atasever, Melike; Koksal, Deniz; Karasartova, Djursun; Kiyan, Mehmet
Use of qPCR for the study of hepatotoxic cyanobacteria population dynamics.
Toxic cyanobacteria blooms are increasingly frequent and object of greater concern due to its ecological and health impacts. One important lack in the toxic cyanobacteria research field is to understand which parameters influence most and how they operate to regulate the overall levels of cyanotoxins in a body of water. MC concentration is believed to be influenced by changes in several seasonal environmental factors that influence the succession of toxic cyanobacteria. In the last years, qPCR (quantitative polymerase chain reaction) has been applied to determine the seasonal and temporal shifts in the proportions of MC-producing and non-MC-producing subpopulations by quantifying both mcy genotypes and total population numbers. We discuss the most prominent and recent studies using qPCR to address hepatotoxic cyanobacteria population dynamics and evaluate how they helped understanding the factors promoting the growth of toxic strains in situ and the succession of hepatotoxin-producing genera in natural populations. PMID:21735229
Martins, António; Vasconcelos, Vitor
Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5? harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets,
Changsoo Lee; Jaai Kim; Seung Gu Shin; Seokhwan Hwang
QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology
The quantitative PCR (QPCR) assay for DNA damage and repair has been used extensively in laboratory species. More recently,\\u000a it has been adapted to ecological settings. The purpose of this article is to provide a detailed methodological guide that\\u000a will facilitate its adaptation to additional species, highlight its potential for ecotoxicological and biomonitoring work,\\u000a and critically review the strengths and
Joel N. Meyer
Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks ( Anas platyrhynchos)
Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan®) probes was employed. It provides a
Shaman Muradrasoli; Nahla Mohamed; Ákos Hornyák; Jan Fohlman; Björn Olsen; Sándor Belák; Jonas Blomberg
Why the need for qPCR publication guidelines?—The case for MIQE
The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate
Stephen A. Bustin
Comparison of Fecal Indicator Bacteria Densities in Marine Recreational Waters by QPCR
The US EPA is currently investigating the use of quantitative PCR (qPCR) analysis techniques to estimate densities of fecal\\u000a indicator bacteria (FIB) in recreational waters. Present water quality guidelines, based on culturable FIB, prevent same day\\u000a water quality determination, whereas results from qPCR-based approaches are available within several hours. Epidemiological\\u000a studies at Publicly-Owned Treatment Works (POTW)-impacted freshwater beaches have also
Eunice C. Chern; Kristen P. Brenner; Larry Wymer; Richard A. Haugland
Efficiency clustering for low-density microarrays and its application to QPCR
BACKGROUND: Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each
Eric F. Lock; Ryan Ziemiecki; J. S. Marron; Dirk P. Dittmer
Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol
BACKGROUND: The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. RESULTS: Harvesting whole cells
Kjetil Hodne; Trude M Haug; Finn-Arne Weltzien
Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.
A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin×tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting CT bias allowed accurate quantitative measurements to be obtained. PMID:24128588
Aldeguer, Miriam; López-Andreo, María; A Gabaldón, José; Puyet, Antonio
Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction
RT-qPCR is the standard method for studying changes in relative transcript level in different experimental and clinical conditions and in different tissues. No validated reference genes have been reported for the normalization of transcript level in platelets. The very low level of platelet RNA and the elimination of leukocyte contamination represented special methodological difficulties. Our aims were to apply a simple technique to separate platelets for transcript level studies, and select the most stable reference genes for platelets from healthy individuals and from patients with the history of myocardial infarction. We developed a simple, straightforward method of platelet separation for RNA isolation. Platelet activation was inhibited by using acid-citrate-dextrose for anticoagulation and by prostaglandin E1. Leukocyte contamination was eliminated by three consecutive centrifugations. Samples prepared by this method were free of leukocytes, showed no inhibition in PCR reaction and no RNA degradation. The assay demands low blood volume, which complies with the requirements of everyday laboratory routine. Seventeen potential reference genes were investigated, but eight of them were excluded during optimization. The stability of the remaining genes, EEF2, EAR, ACTB, GAPDH, ANAPC5, OAZ1, HDGF, GNAS, and CFL1, were determined by four different descriptive statistics. GAPDH, GNAS, and ACTB were shown to be the most stable genes in platelets of healthy individuals, while HDGF, GNAS, and ACTB were the most stable in platelets of patients with the history of myocardial infarction. The results confirm that data normalization needs assessment of appropriate reference genes for a particular sample set.
Zsori, Katalin S.; Muszbek, Laszlo; Csiki, Zoltan; Shemirani, Amir H.
Overestimation of the Abundance of Sulfate-Reducing Bacteria in Human Feces by Quantitative PCR Targeting the Desulfovibrio 16S rRNA Gene ?
The dominant genus of sulfate-reducing bacteria (SRB) in humans is Desulfovibrio, and quantitative PCR (QPCR) targeting the 16S rRNA gene is often used in assays. We show that the 16S rRNA gene assay overestimated SRB abundance in feces from 24 adults compared to QPCR assays using primers targeting two genes involved in SRB energy metabolism.
Christophersen, C. T.; Morrison, M.; Conlon, M. A.
Alignment of gene expression profiles from test samples against a reference database: New method for context-specific interpretation of microarray data
Background Gene expression microarray data have been organized and made available as public databases, but the utilization of such highly heterogeneous reference datasets in the interpretation of data from individual test samples is not as developed as e.g. in the field of nucleotide sequence comparisons. We have created a rapid and powerful approach for the alignment of microarray gene expression profiles (AGEP) from test samples with those contained in a large annotated public reference database and demonstrate here how this can facilitate interpretation of microarray data from individual samples. Methods AGEP is based on the calculation of kernel density distributions for the levels of expression of each gene in each reference tissue type and provides a quantitation of the similarity between the test sample and the reference tissue types as well as the identity of the typical and atypical genes in each comparison. As a reference database, we used 1654 samples from 44 normal tissues (extracted from the Genesapiens database). Results Using leave-one-out validation, AGEP correctly defined the tissue of origin for 1521 (93.6%) of all the 1654 samples in the original database. Independent validation of 195 external normal tissue samples resulted in 87% accuracy for the exact tissue type and 97% accuracy with related tissue types. AGEP analysis of 10 Duchenne muscular dystrophy (DMD) samples provided quantitative description of the key pathogenetic events, such as the extent of inflammation, in individual samples and pinpointed tissue-specific genes whose expression changed (SAMD4A) in DMD. AGEP analysis of microarray data from adipocytic differentiation of mesenchymal stem cells and from normal myeloid cell types and leukemias provided quantitative characterization of the transcriptomic changes during normal and abnormal cell differentiation. Conclusions The AGEP method is a widely applicable method for the rapid comprehensive interpretation of microarray data, as proven here by the definition of tissue- and disease-specific changes in gene expression as well as during cellular differentiation. The capability to quantitatively compare data from individual samples against a large-scale annotated reference database represents a widely applicable paradigm for the analysis of all types of high-throughput data. AGEP enables systematic and quantitative comparison of gene expression data from test samples against a comprehensive collection of different cell/tissue types previously studied by the entire research community.
Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications
BACKGROUND: EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we
Fei Mao; Wai-Yee Leung; Xing Xin
Validation of RT-qPCR reference genes for in planta expression studies in Hemileia vastatrix, the causal agent of coffee leaf rust.
Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions. Reference genes are often used for this purpose, but the stability of their expression levels requires validation under experimental conditions. Moreover, pathogenic fungi undergo important biomass variations along their infection process in planta, which raises the need for an adequate method to further normalise the proportion of fungal cDNA in the total plant and fungus cDNA pool. In this work, the expression profiles of seven reference genes [glyceraldehyde-3-phosphate dehydrogenase (GADPH), elongation factor (EF-1), Beta tubulin (?-tubulin), cytochrome c oxidase subunit III (Cyt III), cytochrome b (Cyt b), Hv00099, and 40S ribosomal protein (40S_Rib)] were analysed across 28 samples, obtained in vitro (germinated uredospores and appressoria) and in planta (post-penetration fungal growth phases). Gene stability was assessed using the statistical algorithms incorporated in geNorm and NormFinder tools. Cyt b, 40S_Rib, and Hv00099 were the most stable genes for the in vitro dataset, while 40S_Rib, GADPH, and Cyt III were the most stable in planta. For the combined datasets (in vitro and in planta), 40S_Rib, GADPH, and Hv00099 were selected as the most stable. Subsequent expression analysis for a gene encoding an alpha subunit of a heterotrimeric G-protein showed that the reference genes selected for the combined dataset do not differ significantly from those selected specifically for the in vitro and in planta datasets. Our study provides tools for correct validation of reference genes in obligate biotrophic plant pathogens, as well as the basis for RT-qPCR studies in H. vastatrix. PMID:21872186
Vieira, Ana; Talhinhas, Pedro; Loureiro, Andreia; Duplessis, Sébastien; Fernandez, Diana; Silva, Maria do Céu; Paulo, Octávio S; Azinheira, Helena Gil
Validation of the ?-amy1 transcription profiling assay and selection of reference genes suited for a RT-qPCR assay in developing barley caryopsis.
Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of ?-amylase (?-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize ?-amy1 analysis for study of the impact of ?-amy1 expression upon barley end-use quality. PMID:22860024
Ovesná, Jaroslava; Ku?era, Ladislav; Vaculová, Kate?ina; Štrymplová, Kamila; Svobodová, Ilona; Milella, Luigi
Validation of the ?-amy1 Transcription Profiling Assay and Selection of Reference Genes Suited for a RT-qPCR Assay in Developing Barley Caryopsis
Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of ?-amylase (?-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize ?-amy1 analysis for study of the impact of ?-amy1 expression upon barley end-use quality.
Ovesna, Jaroslava; Kucera, Ladislav; Vaculova, Katerina; Strymplova, Kamila; Svobodova, Ilona; Milella, Luigi
Discrimination of viable from non-viable gram-negative bacterial pathogens in airborne particles using propidium monoazide-assisted qPCR.
The presence of bacterial pathogens in airborne particulate matter (PM) has been of considerable concern from the public health standpoint. Conventional culture-based methods are tedious, time consuming and are unable to quantify stressed viable but non-culturable (VBNC) populations of these pathogens. This study reports the optimization, validation and application of a new and rapid quantitative method for enumeration of four live potential Gram-negative bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas hydrophila) in PM of biomass burning origin. This method makes use of an intercalating dye (propidium monoazide, PMA) in conjunction with real-time PCR (qPCR) analysis following DNA extraction from PM samples for distinguishing viable from non-viable potential bacterial pathogens. This method was not affected by the complex matrix of the environmental samples, nor by any PCR inhibition effects. The number of viable pathogens ranged from 0 to 8×10(4) gene copies/m(3) in PM. With the exception of A. hydrophilia, all the three pathogens were found to be present in PM. The correlation between the counts obtained using the PMA-qPCR (modified qPCR) and those from the culture-based method was very high with R(2)~1.0 and p value<0.0001. PMID:23428754
Kaushik, Rajni; Balasubramanian, Rajasekhar
A novel procedure for absolute real-time quantification of gene expression patterns
Background Temporal and tissue-specific patterns of gene expression play important roles in functionality of a biological system. Real-time quantitative polymerase chain reaction (qPCR) technique has been widely applied to single gene expressions, but its potential has not been fully released as most results have been obtained as fold changes relative to control conditions. Absolute quantification of transcripts as an alternative method has yet to gain popularity because of unresolved issues. Results We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/?l, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA) qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (Ipomoea purpurea, Nicotiana benthamiana) and tissues (petals and leaves). Conclusion By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells.
Selection and validation of reference genes for real-time RT-PCR studies in the non-model species Delomys sublineatus, an endemic Brazilian rodent.
Quantitative real-time RT-PCR (qRT-PCR) is a sensitive technique for gene expression analysis. A critical factor for creating reliable data in relative quantification is the normalization of the expression data of genes of interest. Therefore the needed normalization factor is calculated out of the expression data of co-amplified genes that are stable expressed in the certain sample material, the so-called reference genes. In this study, we demonstrate the important process of validating potential reference genes using a non-model species. As there are almost no sequences known of the Pallid Atlantic Forest Rat (Delomys sublineatus), a rodent used as indicator species in conservation studies of the endangered Brazilian rainforest, suitable primer sets are more problematic to find than in model species. Out of nine tested primer sets designed for the fully sequenced Mus musculus, five could be used for the establishment of a proper running SYBR-Green assay and validation of their constant expression. qRT-PCR results of 12 cDNAs of Delomys livers were analyzed with three different validation software programs: BestKeeper, NormFinder and geNorm. Our approach showed that out of the five (Sdha, Canx, Pgk1, Actb and Actg1) potential reference genes, the first four should be used for accurate normalization in further relative quantification analyses. Transferring data from close-by model organisms makes high sensitive real-time RT-PCR applicable even to free-ranging non-model organisms. Our approach might be suitable for other non-model organisms. PMID:20059981
Weyrich, Alexandra; Axtner, Jan; Sommer, Simone
Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis
BACKGROUND: Reference genes are used as internal standards to normalize mRNA abundance in quantitative real-time PCR and thereby allow a direct comparison between samples. So far most of these expression studies used human or classical laboratory model species whereas studies on non-model organism under in-situ conditions are quite rare. However, only studies in free-ranging populations can reveal the effects of
Jan Axtner; Simone Sommer
Gene transcription in sea otters (Enhydra lutris); development of a diagnostic tool for sea otter and ecosystem health.
Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real-time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter (Enhydra lutris); sea otter-specific qPCR primer sequences for the genes of interest are defined. We establish a 'reference' range of transcripts for each gene in a group of clinically healthy captive and free-ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno-modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell-cell adhesion. The cycle threshold (C(T)) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment. PMID:21848762
Bowen, Lizabeth; Miles, A Keith; Murray, Michael; Haulena, Martin; Tuttle, Judy; Van Bonn, William; Adams, Lance; Bodkin, James L; Ballachey, Brenda; Estes, James; Tinker, M Tim; Keister, Robin; Stott, Jeffrey L
Evaluation of reference genes for quantitative polymerase chain reaction across life-cycle stages and tissue types of Tribolium castaneum
Technology Transfer Automated Retrieval System (TEKTRAN)
The genome of the genetic model for coleopteran insects, Tribolium castaneum, is now available for downstream applications. To facilitate gene expression studies in T. castaneum, genes were evaluated for suitability in comparisons across tissues and/or developmental stages. In less diverse samples, ...
Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes
Background Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, ?-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes. Results The expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes. Conclusion Generally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.
Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust
Background The Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust. Results Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust. Conclusion Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.
The origins of human gene mapping. With particular reference to the contributions of J. B. S. Haldane.
Methods in human gene mapping, including those developed by Bell and Haldane, Morton, and the more recent ones using somatic cell fusion, restriction fragment length polymorphism (RFLP), and multipoint linkage, are briefly reviewed. PMID:3328738
Dronamraju, K R
Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model
With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent\\u000a need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying\\u000a the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a\\u000a sensitive and highly reproducible technique of gene expression
Lingyan XuXinran; Xinran Ma; Bin Cui; Xiaoying Li; Guang Ning; Shu Wang
Photoelectrocatalytic disinfection of water and wastewater: performance evaluation by qPCR and culture techniques.
Photoelectrocatalytic oxidation (PEC) was evaluated as a disinfection technique using water and secondary treated wastewater spiked with Escherichia coli and Enterococcus faecalis. PEC experiments were carried out using a TiO(2)/Ti-film anode and a zirconium cathode under simulated solar radiation. Bacterial inactivation was monitored by culture and quantitative polymerase chain reaction (qPCR). Inactivation rates were enhanced when the duration of the treatment was prolonged and when the bacterial density and the complexity of the water matrix were decreased. E. coli cells were reduced by approximately 6 orders of magnitude after 15 min of PEC treatment in water at 2V of applied potential and an initial concentration of 10(7) CFU/mL; pure photocatalysis (PC) led to about 5 log reduction, while electrochemical oxidation alone resulted in negligible inactivation. The superiority of PEC relative to PC can be attributed to a more efficient separation of the photogenerated charge carriers. Regarding disinfection in mixed bacterial suspensions, E. coli was more susceptible than E. faecalis at a potential of 2V. The complex composition of wastewater affected disinfection efficiency, yielding lower inactivation rates compared to water treatment. qPCR yielded lower inactivation rates at longer treatment times than culture techniques, presumably due to the fact that the latter do not take into account the viable but not culturable state of microorganisms. PMID:23428546
Venieri, Danae; Chatzisymeon, Efthalia; Politi, Eleonora; Sofianos, Spiridon S; Katsaounis, Alexandros; Mantzavinos, Dionissios
Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei
Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.
Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol
Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with ?-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.
Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment
Background Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment. Results In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*105GU/L while L. pneumophila were detected in a range from LOQ to 6.8*105 GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*106 CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r2 = 0.31 for culture and Legionella spp. assay, r2 = 0.20 for culture and L. pneumophila assay). Conclusion Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.
Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications
Background EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions. Results This study systematically compared the DNA binding profiles of the two dyes under different conditions and had these findings: a) EG had a lower binding affinity for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent preference for either GC- or AT-rich sequence while SG had a slight preference for AT-rich sequence; c) both dyes showed substantially lower affinity toward ssDNA than toward dsDNA and even lower affinity toward shorter ssDNA fragments except that this trend was more pronounced for EG. Our results also demonstrated that EG was stable both under PCR condition and during routine storage and handling. In the comparative qPCR study, both EG and SG exhibited PCR interference when used at high dye concentration, as evident from delayed Ct and/or nonspecific product formation. The problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (>500 bp). However, qPCR using EG tolerated a significantly higher dye concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak. These differences in qPCR performance between the two dyes are believed to be attributable to their differences in DNA binding profiles. Conclusion These findings suggest that an ideal qPCR dye should possess several DNA-binding characteristics, including a "just right" affinity for dsDNA and low or no affinity for ssDNA and short DNA fragments. The favorable DNA-binding profile of EG, coupled with its good stability and instrument-compatibility, should make EG a promising dye for qPCR and related applications.
Mao, Fei; Leung, Wai-Yee; Xin, Xing
Molecular pathology of brain edema after severe burns in forensic autopsy cases with special regard to the importance of reference gene selection.
Brain edema is believed to be linked to high mortality incidence after severe burns. The present study investigated the molecular pathology of brain damage and responses involving brain edema in forensic autopsy cases of fire fatality (n?=?55) compared with sudden cardiac death (n?=?11), mechanical asphyxia (n?=?13), and non-brain injury cases (n?=?22). Postmortem mRNA and immunohistochemical expressions of aquaporins (AQPs), claudin5 (CLDN5), and matrix metalloproteinases (MMPs) were examined. Prolonged deaths due to severe burns showed an increase in brain water content, but relative mRNA quantification, using different normalization methods, showed inconsistent results: in prolonged deaths due to severe burns, higher expression levels were detected for all markers when three previously validated reference genes, PES1, POLR2A, and IPO8, were used for normalization, higher for AQP1 and MMP9 when GAPDH alone was used for normalization and higher for MMP9, but lower for MMP2 when B2M alone was used for normalization. Additionally, when B2M alone was used for normalization, higher expression of AQP4 was detected in acute fire deaths. Furthermore, the expression stability values of these five reference genes calculated by geNorm demonstrated that B2M was the least stable one, followed by GAPDH. In immunostaining, only AQP1 and MMP9 showed differences among the causes of death: they were evident in most prolonged deaths due to severe burns. These findings suggest that systematic analysis of gene expressions using real-time PCR might be a useful procedure in forensic death investigation, and validation of reference genes is crucial. PMID:23702882
Wang, Qi; Ishikawa, Takaki; Michiue, Tomomi; Zhu, Bao-Li; Guan, Da-Wei; Maeda, Hitoshi
Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods: real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR PCR).
Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n=112, bone marrow n=15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism. PMID:23238335
Kletzel, Morris; Huang, Wei; Olszewski, Marie; Khan, Sana
Genetics Home Reference: Stroke
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Genetics Home Reference: Seizures
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Effect of mild restriction of food intake on gene expression profile in the liver of young rats: reference data for in vivo nutrigenomics study.
Recent transcriptomics studies on the effect of long-term or severe energy restriction (ER) have revealed that many genes are dynamically modulated by this condition in rodents. The present study was conducted to define the global gene expression profile in response to mild ER treatment. Growing rats were fed with reduced amount of diet (5-30 % ER) for 1 week or 1 month. Using DNA microarray analysis of the liver, seventy-two genes that were consistently changed through the different ER levels were identified. Many were related to lipid metabolism including genes encoding key enzymes such as carnitine palmitoyltransferase 1 and fatty acid synthase. Interestingly, a number of genes were altered even by 5 % ER for 1 week where no differences in weight gain were observed. The information obtained in the present study can be used as a valuable reference data source in the transcriptomics studies of food and nutrition in which subtle differences in food intake sometimes hinder appropriate interpretation of the data. PMID:20447325
Saito, Kenji; Ohta, Yutaka; Sami, Manabu; Kanda, Tomomasa; Kato, Hisanori
Maternal low-protein diet alters the expression of real-time quantitative polymerase chain reaction reference genes in an age-, sex-, and organ-dependent manner in rat offspring.
Altered perinatal environment, often manifested as low birth weight, is thought to contribute to greater susceptibility for hypertension, hyperlipidemia, and diabetes as a result of epigenetic modifications and alteration of transcriptional activity for key genes. Real-time polymerase chain reaction is a useful technique for the quantitative determination of differences in transcriptional activity. Real-time quantitative polymerase chain reaction data analyses require normalization of transcriptional activity of target genes to an endogenous control, usually a reference gene. In response to reports of altered expression of reference genes in various experimental models, we hypothesized that adverse perinatal environment alters reference gene expression. We examined the expression of the following reference genes in the offspring of a rodent maternal low-protein diet model: ?-actin, hypoxanthine phosphoribosyltransferase 1, TATA-box-binding protein, glyceraldehyde-3-phosphate dehydrogenase, and glucuronidase-? in brain, heart, kidneys, and intestines. We found altered expression in brain, heart, and kidneys for each of the reference genes measured; these effects were age, organ, and sex dependent. Glyceraldehyde-3-phosphate dehydrogenase and glucuronidase-? were found to be the least affected by these variables, whereas hypoxanthine phosphoribosyltransferase 1 was the most inconsistent. Our findings underscore the importance of empirical determination of a reliable reference gene for real-time polymerase chain reaction studies in the low-protein diet model. PMID:23507230
DuBois, Barent; Pearson, Jacob; Hastings, Bonnie; Mahmood, Tahir; Chan, Tammy; Alnakhli, Ali; Cherala, Ganesh
Expressed Sequence Reference Standards for Evaluating Stage-specific Gene Expression in Southern Green Lacewings, Chrysoperla rufilabris
Technology Transfer Automated Retrieval System (TEKTRAN)
Five developmental stages of Chrysoperla rufilabris were tested using nine primer pairs. Three sequences were highly expressed at all life stages and six were differentially expressed. These primer pairs may be used as standards to quantitate functional gene expression associated with physiological ...
Interpretation of the Prostate Cancer Gene 3 in Reference to the Individual Clinical Background: Implications for Daily Practice
Introduction: The prostate cancer gene 3 (PCA3) urine assay for the diagnosis of prostate cancer was introduced into clinical practice at the end of 2006. We report our experience with the test in a routine clinical setting and discuss the interpretation of the test results in the context of the individual patient history. Material and Methods: We retrospectively reviewed the
David Schilling; Jörg Hennenlotter; Marita Munz; Ulf Bökeler; Karl-Dietrich Sievert; Arnulf Stenzl
Identification of Suitable Reference Genes for qRT-PCR Analysis of Circulating microRNAs in Hepatitis B Virus-Infected Patients
Circulating microRNAs (miRNAs) were found to exist in serum\\/plasma in a highly stable, cell-free form, and aberrantly expressed\\u000a in many human diseases. Currently, the expression levels of circulating miRNAs are estimated by quantitative real-time polymerase\\u000a chain reaction. However, no study has systematically evaluated reference genes for evaluating circulating microRNA expression.\\u000a This study describes the identification and characterization of an appropriate
Hai-Tao Zhu; Qiong-Zhu Dong; Guan Wang; Hai-Jun Zhou; Ning Ren; Hu-Liang Jia; Qing-Hai Ye; Lun-Xiu Qin
Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis.
Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed. PMID:22841564
Devonshire, Alison S; Sanders, Rebecca; Wilkes, Timothy M; Taylor, Martin S; Foy, Carole A; Huggett, Jim F
Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization
BACKGROUND: Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare. In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved
Katia Cappelli; Michela Felicetti; Stefano Capomaccio; Giacomo Spinsanti; Maurizio Silvestrelli; Andrea Verini Supplizi
Detection of pathogens in water: from phylochips to qPCR to pyrosequencing.
Waterborne pathogens pose a significant threat to human health and a proper assessment of microbial water quality is important for decision making regarding water infrastructure and treatment investments and eventually to provide early warning of disease, particularly given increasing global disasters associated with severe public health risks. Microbial water quality monitoring has undergone tremendous transition in recent years, with novel molecular tools beginning to offer rapid, high-throughput, sensitive and specific detection of a wide spectrum of microbial pathogens that challenge traditional culture-based techniques. High-density microarrays, quantitative real-time PCR (qPCR) and pyrosequencing which are considered to be breakthrough technologies borne out of the 'molecular revolution' are at present emerging rapidly as tools of pathogen detection and discovery. Future challenges lie in integrating these molecular tools with concentration techniques and bioinformatics platforms for unbiased guide of pathogen surveillance in water and developing standardized protocols. PMID:22153035
Aw, Tiong Gim; Rose, Joan B
Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.
Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring. PMID:23325054
Dittami, Simon M; Hostyeva, Vladyslava; Egge, Elianne Sirnćs; Kegel, Jessica U; Eikrem, Wenche; Edvardsen, Bente
Identification of internal control genes for quantitative polymerase chain reaction in mammary tissue of lactating cows receiving lipid supplements.
Dietary lipid supplements affect mammary lipid metabolism partly through changes in lipogenic gene expression. Quantitative PCR (qPCR) is a sensitive, reliable, and accurate technique for gene expression analysis. However, variation introduced in qPCR data by analytical or technical errors needs to be accounted for via normalization using appropriate internal control genes (ICG). Objectives were to mine individual bovine mammary microarray data on >13,000 genes across 66 cows from 2 independent studies to identify the most suitable ICG for qPCR normalization. In addition to unsupplemented control diets, cows were fed saturated or unsaturated lipids for 21 d or were infused with supplements (butterfat, conjugated linoleic acid mixture, long-chain fatty acids) into the abomasum to modify milk fat synthesis and fatty acid profiles. We identified 49 genes that did not vary in expression across the 66 samples. Subsequent gene network analysis revealed that 22 of those genes were not co-regulated. Among those COPS7A, CORO1B, DNAJC19, EIF3K, EMD, GOLGA5, MTG1, UXT, MRPL39, GPR175, and MARVELD1 (sample/reference expression ratio = 1 +/- 0.1) were selected for PCR analysis upon verification of goodness of BLAT/BLAST sequence and primer design. Relative expression of B2M, GAPDH, and ACTB, previously used as ICG in bovine mammary tissue, was highly variable (0.9 +/- 0.6) across studies. Gene stability analysis via geNorm software uncovered MRPL39, GPR175, UXT, and EIF3K as having the most stable expression ratio and, thus, suitable as ICG. Analysis also indicated that use of 3 ICG was most appropriate for calculating a normalization factor. Overall, the geometric average of MRPL39, UXT, and EIF3K is ideal for normalization of mammary qPCR data in studies involving lipid supplementation of dairy cows. These novel ICG could be used for normalization in similar studies as alternatives to the less-reliable ACTB, GAPDH, or B2M. PMID:19389958
Kadegowda, A K G; Bionaz, M; Thering, B; Piperova, L S; Erdman, R A; Loor, J J
Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS).
Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells. PMID:19797679
Liu, Zhenyi; Obenauf, Anna C; Speicher, Michael R; Kopan, Raphael
Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera
Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEf?1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEf?1 or BoechUBQ and BoechEf?1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEf?1), in anthers from both reproductive systems (BoechACT2 and BoechEf?1), in apomictic vegetative tissues (BoechEf?1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEf?1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEf?1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively) Conclusions From a total of six tested genes, BoechRPS18, BoechEf?1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.
Relationship and Variation of qPCR and Culturable Enterococci Estimates in Ambient Surface Waters Are Predictable
The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based meth...
The U.S. EPA has initiated a new recreational water study to evaluate the correlation between illness rates in swimmers and Enterococcus concentrations determined by the mEI agar membrane filter (MF) method and several new technologies including QPCR analysis. Results of this stu...
Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments -
EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the methodâ??s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM a...
Comparison of Enterococcus qPCR analysis results from fresh and marine waters on two real-tme instruments
The U.S. Environmental Protection Agency (EPA) will be recommending a quantitativ e polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this o...
Evaluation of Real-Time Quantitative Polymerase Chain Reaction (qPCR) to Determine Escherichia coli Concentrations at Two Lake Erie Beaches.
National Technical Information Service (NTIS)
During the recreational seasons of 2006 and 2007, the quantitative polymerase chain reaction (qPCR) method was used to determine Escherichia coli (E. coli) concentrations in samples from two Lake Erie beaches. Results from the qPCR method were compared to...
C. M. Kephart R. N. Bushon
Quantitative polymerase chain (QPCR) reaction using the MIMIC approach to estimate Cryptosporidium parvum oocysts, an intestinal pathogen, in municipal water treatment sludge samples
An accurate estimation of the number of Cryptosporidium parvum oocysts in water treatment plant sludge was determined using the Quantitative Polymerase Chain Reaction (QPCR) method. Approximately 8×106purified viable oocysts were spiked into raw water and treated by conventional water treatment methods. The settled sludge was collected and the DNA extracted. The QPCR Mimic produced two competing products that were 300
P Udeh; J Veenstra; AJ Abraham; GH John
Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications.
The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the ?, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood. PMID:22309221
Moreno, Lilliana I; Tate, Courtney M; Knott, Erika L; McDaniel, Jade E; Rogers, Stephanie S; Koons, Barbara W; Kavlick, Mark F; Craig, Rhonda L; Robertson, James M
Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)
Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5?-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.
Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wolfel, Roman
NSDL National Science Digital Library
As a part of the Ocean Drilling Program (ODP) website, this page provides a list of palynological references related to the Cretaceous Period. These references cover an array of topics including Early Cretaceous gymnosperm pollen, implications of palynofacies on petroleum potential, lignite microfossils, Cretaceous megaspore pollen, microspore pollen and depositional environments.
Ocean Drilling Program, Texas A&M University
ERIC Educational Resources Information Center
|This article presents interesting articles that explore several different areas of reference assessment, including practical case studies and theoretical articles that address a range of issues such as librarian behavior, patron satisfaction, virtual reference, or evaluation design. They include: (1) "Evaluating the Quality of a Chat Service"…
Bivens-Tatum, Wayne
Evaluation of RAD51C as cancer susceptibility gene in a large breast-ovarian cancer patient population referred for genetic testing.
Despite extensive analysis of the BRCA1 and BRCA2 genes, germline mutations are detected in <20% of families with a presumed genetic predisposition for breast and ovarian cancer. Recent literature reported RAD51C as a new breast cancer susceptibility gene. In this study, we report the analysis of 410 patients from 351 unrelated pedigrees. All were referred for genetic testing and we selected families with at least one reported case of ovarian cancer in which BRCA1&2 mutations were previously ruled out. We analyzed the coding exons, intron-exons boundaries, and UTRs of RAD51C. Our mutation analysis did not reveal any unequivocal deleterious mutation. In total 12 unique sequence variations were identified of which two were novel. Our study and others suggest a low prevalence of RAD51C mutations with an exception for some founder populations. This observation is in favor of the rare allele hypothesis in the debate over the nature of the genetic contribution to individual susceptibility to breast and ovarian cancer and further genome-wide studies in high risk families are warranted. PMID:22370629
De Leeneer, K; Van Bockstal, M; De Brouwer, S; Swietek, N; Schietecatte, P; Sabbaghian, N; Van den Ende, J; Willocx, S; Storm, K; Blaumeiser, B; Van Asperen, C J; Wijnen, J T; Leunen, K; Legius, E; Michils, G; Matthijs, G; Blok, M J; Gomez-Garcia, E; De Paepe, A; Tischkowitz, M; Poppe, B; Claes, K
Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains
Background The presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome. Here we investigate the extent of polymorphism in eight genes in the mce1 and mce4 operons of M. tuberculosis from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform. Results We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of mce1 and mce4 operons revealed that yrbE1A [Rv0167] was most polymorphic in mce1 operon while yrbE4A [Rv3501c] and lprN [Rv3495c] had the highest number of SNPs in the mce4 operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to M. tuberculosis using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation. Conclusions Our results show that SNPs in the coding sequences of mce1 and mce4 operons in clinical isolates can be significantly high. Moreover, mce4 operon is significantly more polymorphic than mce1 operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in mce1 operon and synonymous substitutions are more in mce4 operon. In silico modeling predict that nonsynonymous SNP at mce1A [Rv0169], a virulence gene could play a pivotal role in causing functional changes in M. tuberculosis that may reflect upon the biology of the bacteria.
Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods
Background Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Results Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Conclusion Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.
Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).
Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale. PMID:22349594
Hornyák, Akos; Bálint, Adám; Farsang, Attila; Balka, Gyula; Hakhverdyan, Mikhayil; Rasmussen, Thomas Bruun; Blomberg, Jonas; Belák, Sándor
[Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors].
The detection of specific nucleic acid (NA) sequences by PCR has revolutionized the biological and medical sciences. Real-time PCR (qPCR) opened up the possibility of obtaining quantitative results. NA extraction is a decisive step prior to qPCR since it may produce either the removal or co-extraction of inhibitory substances of the enzymatic reaction, which in turn affects the amplification efficiency. In the present work we compared the commercial NA extraction kits from Qiagen, Invitrogen and Macherey-Nagel, which were used to extract DNA from mice blood artificially infected with Trypanosoma cruzi and PP7 RNA, Pseudomonas aeruginosa bacteriophage, in spiked aqueous matrices. NA recovery efficiency in samples without inhibitors was similar for the three extraction kits. However, the Invitrogen kit was the only one that remained unaffected in the presence of inhibitors in the samples. PMID:23102460
Poma, Hugo R; Davies, Carolina; Gutiérrez Cacciabue, Dolores; Mora, María C; Basombrío, Miguel Á; Rajal, Verónica B
Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples.
An in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification of bla(NDM-1) DNA was linear over 10 log dilutions (r(2) = 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other ?-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 10(1) to 3 × 10(1) CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 10(1) to 4 × 10(3) CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 10(1) CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of the bla(NDM-1) qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time for bla(NDM-1) detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts. PMID:21690281
Naas, Thierry; Ergani, Ayla; Carrër, Amélie; Nordmann, Patrice
Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8.
Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10 mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200 ng/?L). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (?C(T)) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring. PMID:21256890
Schriewer, A; Wehlmann, A; Wuertz, S
Identification of accurate reference genes for RT-qPCR analysis of formalin-fixed paraffin-embedded tissue from primary non-small cell lung cancers and brain and lymph node metastases.
Lung cancer is the most common cause of cancer-related deaths worldwide, and metastatic spread of the cancer rather than the primary tumor is the main cause of death. However, the molecular alterations of cancer cells leading to the formation of metastasis are poorly understood. This is partly a result of most solid tumor samples available for retrospective studies being archived as formalin-fixed paraffin-embedded (FFPE) specimens causing the nucleic acids to be highly degraded. Furthermore, stably expressed reference genes for normalization of gene expression data using reverse transcriptase quantitative PCR (RT-qPCR) have not been identified for combined analysis of primary lung tumors and the tissues where to the cancer metastasize. Using an optimized RT-qPCR workflow we have analyzed the expression of 23 candidate reference genes in a total of 54 FFPE specimens derived from primary Non-Small Cell Lung Cancer tumors, brain metastases, and lymph node metastases as well as normal lung, lymph node, and brain tissues. We show that every aspect of the workflow is highly reproducible, and the PUM1, TBP, and IPO8 genes were identified as the most stably expressed reference genes among the candidates, by using the GeNorm and NormFinder software programs. Furthermore, we demonstrate that commonly used reference genes such as ACTB (?-actin), GAPDH, and rRNA18S are less stably expressed in the studied samples. The presented workflow and the identified reference genes may facilitate more reliable gene expression studies in lung cancer using RNA from FFPE tissues. PMID:23643276
Sřes, Signe; Sřrensen, Brita Singers; Alsner, Jan; Overgaard, Jens; Hager, Henrik; Hansen, Lise Lotte; Kristensen, Lasse Sommer
Characterization and Quantitation of a Novel ?-Lactamase Gene Found in a Wastewater Treatment Facility and the Surrounding Coastal Ecosystem?
Wastewater treatment plants (WWTPs) are engineered structures that collect, concentrate, and treat human waste, ultimately releasing treated wastewater into local environments. While WWTPs efficiently remove most biosolids, it has been shown that many antibiotics and antibiotic-resistant bacteria can survive the treatment process. To determine how WWTPs influence the concentration and dissemination of antibiotic-resistant genes into the environment, a functional metagenomic approach was used to identify a novel antibiotic resistance gene within a WWTP, and quantitative PCR (qPCR) was used to determine gene copy numbers within the facility and the local coastal ecosystem. From the WWTP metagenomic library, the fosmid insert contained in one highly resistant clone (MIC, ?416 ?g ml?1 ampicillin) was sequenced and annotated, revealing 33 putative genes, including a 927-bp gene that is 42% identical to a functionally characterized ?-lactamase from Staphylococcus aureus PC1. Isolation and subcloning of this gene, referred to as blaM-1, conferred ampicillin resistance to its Escherichia coli host. When normalized to volume, qPCR showed increased concentrations of blaM-1 during initial treatment stages but 2-fold-decreased concentrations during the final treatment stage. The concentration ng?1 DNA increased throughout the WWTP process from influent to effluent, suggesting that blaM-1 makes up a significant proportion of the overall genetic material being released into the coastal ecosystem. Average discharge was estimated to be 3.9 × 1014 copies of the blaM-1 gene released daily into this coastal ecosystem. Furthermore, the gene was observed in all sampled coastal water and sediment samples surrounding the facility. Our results suggest that WWTPs may be a pathway for the dissemination of novel antibiotic resistance genes into the environment.
Uyaguari, Miguel I.; Fichot, Erin B.; Scott, Geoffrey I.; Norman, R. Sean
Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene.
Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers. PMID:16297882
Traverso, Monica; Malnati, Mauro; Minetti, Carlo; Regis, Stefano; Tedeschi, Silvana; Pedemonte, Marina; Bruno, Claudio; Biassoni, Roberto; Zara, Federico
Relationship and variation of qPCR and culturable Enterococci estimates in ambient surface waters are predictable.
The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P < 0.05) at most sites. A linearly decreasing variance of CCE with increasing CFU levels was significant (P < 0.05) or evident for all sites. Both marine and freshwater sites under continuous influence of point-source contamination tended to reveal a relatively constant proportion of CCE to CFU. The consistency in the mean and variance patterns of CCE versus CFU indicates that the relationship of results based on these two methods is more predictable at high CFU levels (e.g., log(10)CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors. PMID:20527919
Whitman, Richard L; Ge, Zhongfu; Nevers, Meredith B; Boehm, Alexandria B; Chern, Eunice C; Haugland, Richard A; Lukasik, Ashley M; Molina, Marirosa; Przybyla-Kelly, Kasia; Shively, Dawn A; White, Emily M; Zepp, Richard G; Byappanahalli, Muruleedhara N
On Referring and Not Referring
Even though it's based on a bad argument, there's something to Strawson's dictum. He might have likened 'referring expression' to phrases like 'eating utensil' and 'dining room': just as utensils don't eat and dining rooms don't dine, so, he might have argued, expressions don't refer. Actually, that wasn't his argument, though it does make you wonder. Rather, Strawson exploited the
KENT BACH; P. F. Strawson
A cross comparison of QPCR to agar-based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs
Molecular methods such as quantitative, real-time polymerase chain reaction (QPCR) are intended to shorten the period between sampling and publicly available results. Cross comparison studies in Racine, WI, USA evaluated QPCR against agar-based (US EPA Method 1600) and defined substrate (IDEXX Colilert-18®) methods for the detection and quantification of Escherichia coli and enterococci in a variety of aqueous environments (wastewater,
Jennifer S. Lavender; Julie L. Kinzelman
Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods.
Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (?CT and ??CT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples. PMID:23123048
Cao, Yiping; Sivaganesan, Mano; Kinzelman, Julie; Blackwood, A Denene; Noble, Rachel T; Haugland, Richard A; Griffith, John F; Weisberg, Stephen B
A strategy for designing multi-taxa specific reference gene systems. example of application--ppi phosphofructokinase (ppi-PPF) used for the detection and quantification of three taxa: maize (Zea mays), cotton (Gossypium hirsutum) and rice (Oryza sativa).
In the first part of the paper, we report the description of a new strategy for the development of a plant reference gene system that can be used for genetically modified organism (GMO) analysis. On the basis of in silico research for candidate genes, the design of degenerate primers allowed the obtention of genomic sequences of the selected gene ppi-phosphofructokinase ( ppi-PPF) for nine taxa in which GMOs have been developed. The comparison and the analysis of inter- and intraspecies sequence variability were performed using a large number of species and cultivars. As an example of application following the detection of single nucleotide polymorphism, we designed specific conventional and real-time polymerase chain reaction tests for the detection and quantification of three taxa, namely, maize, cotton, and rice. This system was highly specific and sensitive. The gene copy number conservation among different cultivars was analyzed and confirmed with a sequencing step. This reference gene system is adequate for use in routine assays for the quantification of GMOs. We then explain briefly the constraints faced and propose recommendations when designing a reference gene system depending on the species to be targeted. PMID:17824661
Chaouachi, Maher; Giancola, Sandra; Romaniuk, Marcel; Laval, Valérie; Bertheau, Yves; Brunel, Dominique
NASA Astrophysics Data System (ADS)
In most electrochemical measurements, it is necessary to keep one of the electrodes in an electrochemical cell at a constant potential. This so-called reference electrode allows control of the potential of a working electrode (e.g. in voltammetry) or the measurement of an indicator electrode (e.g. in potentiometry, see Chap. II.9). The standard hydrogen electrode plays the role of a basic reference element in electrochemical devices; however, in practice, it is difficult to handle. Therefore, secondary reference electrodes are preferred in most experiments. A secondary reference electrode must fulfil the following criteria: (i) it should be chemically and electrochemically reversible, i.e. its potential is governed by the Nernst equation and does not change in time; (ii) the potential must remain almost constant when a small current passes through the electrode and reverse to its original value after such small current flow (i.e. a non-polarisable electrode); and (iii) the thermal coefficient of potential should be small.
Kahlert, Heike
RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts
BACKGROUND: The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR. FINDINGS: The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression
Joëlle Vermeulen; Stefaan Derveaux; Steve Lefever; Els De Smet; Katleen De Preter; Nurten Yigit; Anne De Paepe; Filip Pattyn; Frank Speleman; Jo Vandesompele
Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.
Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3) CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China. PMID:23077478
Meng, Shuang; Xu, Jianguo; Xiong, Yanwen; Ye, Changyun
Gene expression specificity of the mussel antifungal mytimycin (MytM)
We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR
Molruedee Sonthi; Franck Cantet; Mylčne Toubiana; Maria-Rosa Trapani; Maria-Giovanna Parisi; Matteo Cammarata; Philippe Roch
A new molecular approach based on qPCR for the quantification of fecal bacteria in contaminated marine sediments.
Harbour sediments are periodically subjected to dredging operations and their management is mainly based on the assessment of the chemical contamination levels, but the potential risks posed by the presence of pathogenic microorganisms have been largely neglected. Here we first developed new molecular protocols based on the use of Real Time Quantitative PCR (qPCR), targeting both bacterial DNA and the transcription product (rRNA), for the identification and quantification of bacteria of fecal origin (Escherichia coli, Enterococcus spp. and Salmonella spp.) in contaminated harbour sediments. Then, we assessed the effects of bioremediation treatments, conventionally utilized for abating the hydrocarbon contamination in the sediment, on the abundance of fecal bacteria (FB). The qPCR technique was highly specific, sensitive and reproducible, and detected a number of fecal bacteria significantly higher than the classical cultivation techniques. Sediments subjected to bioremediation experiments by biostimulation with inorganic nutrients at different temperatures displayed a significant increase of the abundance of E. coli and Enterococcus spp. These findings suggest the risk of a potential increase of the contamination by pathogenic microorganisms of fecal origin during bioremediation and, as such, highlight the importance of careful monitoring this biological component in harbour sediments when subjected to bio-treatments. PMID:21839120
Luna, Gian Marco; Dell'Anno, Antonio; Pietrangeli, Biancamaria; Danovaro, Roberto
Use of real-time QPCR in biokinetics and modeling of two different ammonia-oxidizing bacteria growing simultaneously.
A real-time quantitative polymerase chain reaction (QPCR) was used to evaluate biokinetic coefficients of Nitrosomonas nitrosa and N. cryotolerans clusters growing simultaneously in a batch mode of ammonia oxidation. The mathematical models based on Monod equation were employed to describe the competitive relationship between these clusters and were fitted to experimental data to obtain biokinetic values. The maximum growth rates (?(m)), half-saturation coefficients (K(S)), microbial yields (Y) and decay coefficients (k(d)) of N. nitrosa and N. cryotolerans were 1.77 and 1.21 day(-1), 23.25 and 23.06 mg N·L(-1), 16 × 10(8) and 1 × 10(8) copies·mg N(-1), 0.26 and 0.20 day(-1), respectively. The estimated coefficients were applied for modeling continuous operations at various hydraulic retention times (HRTs) with an influent ammonia concentration of 300 mg N·L(-1). Modeling results revealed that ammonia oxidation efficiencies were achieved 55-98 % at 0.8-10 days HRTs and that the system was predicted to be washed out at HRT of 0.7 days. Overall, use of QPCR for estimating biokinetic coefficients of the two AOB cluster growing simultaneously by use of ammonia were successful. This idea may open a new direction towards biokinetics of ammonia oxidation in which respirometry tests are usually employed. PMID:23832436
Cho, Kyungjin; Nguyen, Duong Xuan; Lee, Seungyong; Hwang, Seokhwan
Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation-emission matrix spectroscopy and PARAFAC.
Assays that utilize PCR offer powerful tools to detect pathogens and other microorganisms in environmental samples. However, PCR inhibitors present in nucleic acid extractions can increase a sample's limit of detection, skew calculated marker concentrations, or cause false-negative results. It would be advantageous to predict which samples contain various types and levels of PCR inhibitors, especially the humic and fulvic acids that are frequently cited as PCR inhibitors in natural water samples. This study investigated the relationships between quantitative PCR (qPCR) inhibition and the humic and fulvic content of dissolved organic matter (DOM), as well as several other measures of DOM quantity and quality, in water samples. QPCR inhibition was also compared to water quality parameters, precipitation levels, and land use adjacent to the sampling location. Results indicate that qPCR inhibition in the tested water samples was correlated to several humic substance-like, DOM components, most notably terrestrially-derived, humic-like DOM and microbially-derived, fulvic-like DOM. No correlation was found between qPCR inhibition and water quality parameters or land use, but a relationship was noted between inhibition and antecedent rainfall. This study suggests that certain fractions of humic substances are responsible for PCR inhibition from temperate, freshwater systems. PARAFAC modeling of excitation-emission matrix spectroscopy provides insight on the components of the DOM pool that impact qPCR success and may be useful in evaluating methods to remove PCR inhibitors present in samples. PMID:23601829
Gentry-Shields, Jennifer; Wang, Angela; Cory, Rose M; Stewart, Jill R
A mixture model with a reference-based automatic selection of components for disease classification from protein and/or gene expression levels
Background Bioinformatics data analysis is often using linear mixture model representing samples as additive mixture of components. Properly constrained blind matrix factorization methods extract those components using mixture samples only. However, automatic selection of extracted components to be retained for classification analysis remains an open issue. Results The method proposed here is applied to well-studied protein and genomic datasets of ovarian, prostate and colon cancers to extract components for disease prediction. It achieves average sensitivities of: 96.2 (sd = 2.7%), 97.6% (sd = 2.8%) and 90.8% (sd = 5.5%) and average specificities of: 93.6% (sd = 4.1%), 99% (sd = 2.2%) and 79.4% (sd = 9.8%) in 100 independent two-fold cross-validations. Conclusions We propose an additive mixture model of a sample for feature extraction using, in principle, sparseness constrained factorization on a sample-by-sample basis. As opposed to that, existing methods factorize complete dataset simultaneously. The sample model is composed of a reference sample representing control and/or case (disease) groups and a test sample. Each sample is decomposed into two or more components that are selected automatically (without using label information) as control specific, case specific and not differentially expressed (neutral). The number of components is determined by cross-validation. Automatic assignment of features (m/z ratios or genes) to particular component is based on thresholds estimated from each sample directly. Due to the locality of decomposition, the strength of the expression of each feature across the samples can vary. Yet, they will still be allocated to the related disease and/or control specific component. Since label information is not used in the selection process, case and control specific components can be used for classification. That is not the case with standard factorization methods. Moreover, the component selected by proposed method as disease specific can be interpreted as a sub-mode and retained for further analysis to identify potential biomarkers. As opposed to standard matrix factorization methods this can be achieved on a sample (experiment)-by-sample basis. Postulating one or more components with indifferent features enables their removal from disease and control specific components on a sample-by-sample basis. This yields selected components with reduced complexity and generally, it increases prediction accuracy.
Expression profiling of a high-fertility mouse line by microarray analysis and qPCR
BACKGROUND: In a recent study it was demonstrated that a largely increased ovulation number is responsible for high prolificacy in two mouse lines selected for fertility performance. The objective of the present study was to identify genes that are involved in increasing the ovulation number in one of these lines, FL1. For differential expression profiling, ovaries of FL1 and of
Jens Vanselow; Gerd Nürnberg; Dirk Koczan; Martina Langhammer; Hans-Jürgen Thiesen; Norbert Reinsch
A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials
This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for\\u000a genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative\\u000a determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples.\\u000a The paper describes a
Pingjian Deng; Dongyan Yang; Yongcun Yang; Xiaoke Yang; Liangrang Guo; Xiangyang Zhou; Xueling Wang
SYBR ® Green qPCR screening methods for the presence of "35S promoter" and "NOS terminator" elements in food and feed products
The Cauliflower Mosaic Virus "35S promotor" (p35S) and the Agrobacterium "Nopaline Synthase" terminator (tNOS) are the most represented generic recombinant elements in commercial genetically modified\\u000a crops to date. A set of four new SYBR®Green qPCR methods targeting the "p35S" and "tNOS" core elements have been developed. These qPCR methods generate short amplicons\\u000a of 147 and 75 bp for the "p35S" element
Elodie Barbau-PiednoirAntoon; Antoon Lievens; Guillaume Mbongolo-Mbella; Nancy Roosens; Myriam Sneyers; Amaya Leunda-Casi; Marc Van den Bulcke
Quantifying Genes and Transcripts To Assess the In Situ Physiology of \\
Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of \\
Patrick K. H. Lee; Tamzen W. Macbeth; Kent S. Sorenson; Rula A. Deeb; Lisa Alvarez-Cohen
Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves
BackgroundReal-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR
Jens Magnus Bernth Jensen; Mikkel Steen Petersen; Marc Stegger; Lars J. Řstergaard; Bjarne K. Mřller; M. Thomas P. Gilbert
Simplex and duplex polymerase chain reaction analysis of Herculex RW (59122) maize based on one reference molecule including separated fragments of 5' integration site and endogenous gene.
Reference molecules, as positive controls and calibrators, have been recently developed in genetically modified organism analysis as a potential substitute for reference materials derived from plant raw materials. In this study, a novel reference molecule p59122, including the revealed 5' integration sequence of maize Herculex RW (59122), was constructed that was suitable for simplex and duplex event-specific qualitative and quantitative PCR detections. The LOD values were 10 copies both for simplex and duplex qualitative PCR when p59122 was used as the calibrator. These values were comparable to those of using genomic DNA samples with 0.01 and 0.05%, approximately 5 and 25 hyploid genomic DNA copies, respectively. The absolute LOD and LOQ values were confirmed to be as low as 10 and 25 copies of p59122 DNA both in simplex and duplex quantitative systems. Furthermore, ideal quantification data with low bias, SD and RSD values were obtained from the practical samples analyses in simplex and duplex real-time PCR systems using the reference molecule p59122 as a calibrator. All these results suggested that the developed reference molecule p59122 and the qualitative and quantitative PCR detection methods are suitable for identification and quantification of GM maize 59122 and its derived products. PMID:19916386
Li, Xiang; Yang, Litao; Zhang, Jianzhong; Wang, Shu; Shen, Kailin; Pan, Liangwen; Zhang, Dabing
Validation of RT-qPCR reference genes for in planta expression studies in Hemileia vastatrix, the causal agent of coffee leaf rust
Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although
Ana Vieira; Pedro Talhinhas; Andreia Loureiro; Sébastien Duplessis; Diana Fernandez; Maria do Céu Silva; Octávio S. Paulo; Helena Gil Azinheira
Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis : analysis of clinical isolates and standard reference strains
Background The presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes\\u000a in the genome.\\u000a \\u000a \\u000a Here
Rashmi Pasricha; Amita Chandolia; Prija Ponnan; Neeraj Kumar Saini; Sangeeta Sharma; Madhu Chopra; Mandira Varma Basil; Vani Brahmachari; Mridula Bose
Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time qPCR Analysis of Enterococci in Recreational Waters
In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laborator...
Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements
The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for en...
Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments - poster
The U.S. Environmental Protection Agency (EPA) will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this or ...
Selection of reliable reference genes for qRT-PCR studies on cetacean fibroblast cultures exposed to OCs, PBDEs, and 17beta-estradiol.
Quantitative real-time PCR (qRT-PCR) represents an effective molecular technique for the detection of mRNA expression in biological samples. Its sensitivity allows the quantification of slight changes in the regulation of gene transcription but is strictly dependent upon the method followed during the normalization procedure. Relative quantification determines changes in the steady-state mRNA levels of genes across multiple samples and it is assessed by comparison with the levels of one or more internal control RNA. In this context, the choice of constitutively expressed control genes, whose transcription is not affected by the contaminants, appears to be fundamental for the reliability of this technique. During this study, fibroblast cell cultures originated from integumentum biopsies, sampled in the cetacean species Stenella coeruleoalba, have been exposed for 6h to increasing concentrations of different mixtures of compounds with endocrine disruptor capacities (EDCs): organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), and 17beta-estradiol. Ten common housekeeping genes have been tested for the expression of their transcripts in exposed cell cultures using qRT-PCR assays and raw data were analyzed with the two Excel applets geNorm and NormFinder. The genes encoding for SDHA, GAPDH and YWHAZ appear to be the most reliable controls, respectively, for the OC, PBDE and 17beta-estradiol treatments. These results clearly show that the transcription of even widely diffused control genes can be regulated by different treatments and underlie the importance of a careful selection of the optimal housekeeping genes in toxicological studies. PMID:18339435
Spinsanti, Giacomo; Panti, Cristina; Bucalossi, Daniela; Marsili, Letizia; Casini, Silvia; Frati, Francesco; Fossi, Maria Cristina
Quantification of Yersinia enterocolitica in raw milk using qPCR.
This report describes a new, sensitive and specific protocol for rapid detection and quantification of Yersinia enterocolitica in artificially contaminated raw milk samples. The new method is based on an optimized real-time PCR protocol with a TaqMan probe. The primers and probe are based on the chromosomal ail gene. This method was successful for both intended uses: (1) direct detection and quantification of Y. enterocolitica in artificially and naturally contaminated raw milk samples and (2) characterization of growth potential of different serotypes of Y. enterocolitica in raw milk at the most commonly used storage temperatures. The recent method eliminates the pre-PCR enrichment step, which makes it possible to quickly assess milk-related consumer exposure to this pathogen. PMID:22841511
Najdenski, Hristo; Heyndrickx, Marc; Herman, Lieve; Werbrouck, Hadewig; Van Coillie, Els
Evaluation of Real-Time Quantitative Polymerase Chain Reaction (qPCR) to Determine Escherichia coli Concentrations at Two Lake Erie Beaches
During the recreational seasons of 2006 and 2007, the quantitative polymerase chain reaction (qPCR) method was used to determine Escherichia coli (E. coli) concentrations in samples from two Lake Erie beaches. Results from the qPCR method were compared to those obtained by traditional culturing on modified mTEC agar. Regression analysis showed strong, statistically significant correlations between results from the two methods for both years. Correlation coefficients at Edgewater and Villa Angela Beaches were 0.626 and 0.789 for 2006 and 0.667 and 0.829 for 2007, respectively. Linear regression analyses were done to determine how well E. coli concentrations could have been predicted from qPCR results. These hypothetical predictions were compared to the current practice of determining recreational water quality from E. coli concentrations determined for samples collected on the previous day. The qPCR method resulted in a greater percentage of correct predictions of water-quality exceedances than the current method for both beaches and both years. However, because regression equations differed somewhat between both sites and both years, the study did not result in any single relation reliable enough to use for actual real-time prediction of water-quality exceedances for either beach; therefore, a posterior analysis of data was done. Additional years of data may be needed to develop such a relation. Results from this study support the continued development and testing of a qPCR method for providing rapid and accurate estimates of E. coli concentrations for monitoring recreational water quality.
Kephart, Christopher M.; Bushon, Rebecca N.
Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill
BACKGROUND: Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative
Márcia R de Almeida; Carolina M Ruedell; Felipe K Ricachenevsky; Raul A Sperotto; Giancarlo Pasquali; Arthur G Fett-Neto
Reference Frames and Relativity.
ERIC Educational Resources Information Center
Stresses the importance of a reference frame in mechanics. Shows the Galilean transformation in terms of relativity theory. Discusses accelerated reference frames and noninertial reference frames. Provides examples of reference frames with diagrams. (YP)
Swartz, Clifford
NSDL National Science Digital Library
Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.
Excellence, Access
Responses to water withdrawal of tobacco plants genetically engineered with the AtTPS1 gene: a special reference to photosynthetic parameters
We have previously obtained several lines of tobacco transformed with a trehalose-6-phosphate synthase gene of plant origin\\u000a (Arabidopsis thaliana), involved in the first step of the biosynthesis of trehalose, a known osmoprotectant. Two showed distinct intensity of expression:\\u000a high (B5H) and low (B1F). Such lines were analyzed for trehalose-6-phosphate content and the obtained results demonstrated\\u000a to be in accordance with
André M. Almeida; Anabela B. Silva; Susana S. Araújo; Luís A. Cardoso; Dulce M. Santos; José M. Torné; Jorge M. Silva; Matthew J. Paul; Pedro S. Fevereiro
Gene expression patterns in hypoxic and post-hypoxic adult rat retina with special reference to the NMDA receptor and its interactome
Purpose: A gene expression analysis of hypoxic rat retina was undertaken to gain a deeper understanding of the possible molecular mechanisms that underlie hypoxia-induced retinal pathologies and identify possible therapeutic targets. Methods: Rats were made severely hypoxic (6%-7% O2) for 3 h. Some rats were sacrificed at this time, and others were allowed to recover for 24 h under normoxic
Lori Anne Crosson; Roger A. Kroes; Joseph R. Moskal; Robert A. Linsenmeier
Evaluation of the state of the gene pool of natural populations dwelling in the fragmented landscape of Moscow and Moscow region (with special reference to brown frogs)
A system of urban ecological genetic monitoring of vertebrates has been developed for the first time in Russia and in the\\u000a world. As a model, two species of brown frogs, Rana arvalis Nilss. and R. temporaria L. (16 populations, seven isozyme loci) were used. An evaluation of the gene pool state in urban frog populations has shown\\u000a that the diversity
V. M. Makeeva; M. M. Belokon; O. P. Malyuchenko; O. A. Leont'eva
Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes
Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...
Molecular Identification and Real-time Quantitative PCR (qPCR) for Rapid Detection of Thelohanellus kitauei, a Myxozoan Parasite Causing Intestinal Giant Cystic Disease in the Israel Carp
Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.
Seo, Jung Soo; Jeon, Eun Ji; Kim, Moo Sang; Woo, Sung Ho; Kim, Jin Do; Jung, Sung Hee; Park, Myoung Ae; Jee, Bo Young; Kim, Jin Woo; Kim, Yi-Cheong
Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp
In this report, real-time quantitative PCR (TaqMan® qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic\\u000a marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable,\\u000a microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean\\u000a Lithistid sponges have shown a wide diversity of
M. Cassler; C. L. Peterson; A. Ledger; S. A. Pomponi; A. E. Wright; R. Winegar; P. J. McCarthy; J. V. Lopez
Detection and Quantitation of E. faecalis by Real-time PCR (qPCR), Reverse Transcription-PCR (RT-PCR), and Cultivation During Endodontic Treatment
Enterococcus faecalis is frequently recovered from refractory endodontic infections and has been implicated in endodontic treatment failures. This study compared real-time quantitative PCR (qPCR) assay to cultivation for E. faecalis detection and quantitation during endodontic treatment. A reverse-transcription PCR (RT-PCR) assay was also developed to detect the bacterium clinically in the viable but nonculturable (VBNC) state. Intra-canal samples (n =
John M. Williams; Martin Trope; Daniel J. Caplan; Diane C. Shugars
Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons
BACKGROUND: Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV\\/HHV8) have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR) assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2) lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF
A Gregory Bruce; Angela M Bakke; Margaret E Thouless; Timothy M Rose
Quantification of Airborne Influenza and Avian Influenza Virus in a Wet Poultry Market using a Filter\\/Real-time qPCR Method
Influenza viruses cause pandemics in humans. The aim of this study was therefore to evaluate filter\\/real-time qPCR to quickly and accurately determine and quantify the airborne influenza and avian influenza virus. In this study, the sampling stress of filtration to influenza virus and the storage effects for both the virus and extracted RNA were evaluated in the laboratory. Then, the
Pei-Shih Chen; Chien Kun Lin; Feng Ta Tsai; Chun-Yuh Yang; Chien-Hung Lee; Yen-Shung Liao; Ching-Ying Yeh; Chwan-Chuen King; Jiunn-Lin Wu; Yu-Chun Wang; Kuei-Hsiang Lin
Contamination level and location of recreational freshwater influence the ability to predict Escherichia coli concentration by qPCR targeting Bacteroides.
Fecal bacteria are common microbial contaminants in freshwater with the potential to cause human illness. Detection of these microbes have traditionally relied on microbial plating to enumerate colonies of fecal indicator bacteria (FIB) such as Escherichia coli (E. coli), which can take 24 h or longer to complete. Quantitative PCR (qPCR) is a rapid and sensitive method for detection of FIB in recreational water that could compliment or potentially substitute for microbial plating. In this study, we have isolated DNA from the beach water on the shoreline at three different locations of Lake Erie and subjected these samples to qPCR to examine the relative abundance of Bacteroides. These values were compared to colony forming units (CFU) of E. coli. The resultant linear regressions between these different measurements of microbe concentration were used to determine the efficacy of qPCR targeting Bacteroides at predicting E. coli concentrations that are relevant for decision making by recreational water managers. Our findings indicate that the ability of Bacteroides to serve as an early predictive tool for E. coli CFU concentration depends on sample location and level of bacterial contamination, but can be used in some cases to supplement recreational water quality measurement and consequential management. PMID:22466662
Mulugeta, Surafel; Hindman, Ryan; Olszewski, Adam M; Hoover, Kaitlyn; Greene, Kendall; Lieberman, Matthew; Mauro, Steven A
Cloning of the human dopamine D5 receptor gene and identification of a highly polymorphic microsatellite for the DRD5 locus that shows tight linkage to the chromosome 4p reference marker RAF1P1
The authors identified a cosmid clone with exact sequence homology to part of the human dopamine D5 receptor gene (DRD5) after screening a cosmid library with the human DRD1 gene. The dopamine D5 receptor was mapped to chromosome 4p15.1-p15.3 by in situ hybridization and using a somatic cell hybrid panel. They report here the further localization of the DRD5 gene following identification of a highly polymorphic dinucleotide repeat sequence in the cosmid clone. The microsatellite (D5(CT/GT/GA)[sub n]) had 12 alleles with a polymorphic information content value of 0.77. Linkage analysis in 39 CEPH pedigrees demonstrated tight linkage to the chromosome 4p reference marker RAF1P1 (Z[sub maxf] 20.66 at [theta][sub f] 0.05 and Z[sub maxM] 16.57 at [theta][sub m] 0.07). 16 refs., 2 tabs.
Sherrington, R.; Mankoo, B.; Kalsi, G.; Gurling, H.; Curtis, D. (Univ. College London Medical School (United Kingdom)); Attwood, J.; Povey, S. (Galton Lab., London (United Kingdom)); Buetow, K. (Fox Chase Cancer Center, Philadelphia, PA (United States))
OakContigDF159.1, a reference library for studying differential gene expression in Quercus robur during controlled biotic interactions: use for quantitative transcriptomic profiling of oak roots in ectomycorrhizal symbiosis.
Oaks (Quercus spp.), which are major forest trees in the northern hemisphere, host many biotic interactions, but molecular investigation of these interactions is limited by fragmentary genome data. To date, only 75 oak expressed sequence tags (ESTs) have been characterized in ectomycorrhizal (EM) symbioses. We synthesized seven beneficial and detrimental biotic interactions between microorganisms and animals and a clone (DF159) of Quercus robur. Sixteen 454 and eight Illumina cDNA libraries from leaves and roots were prepared and merged to establish a reference for RNA-Seq transcriptomic analysis of oak EMs with Piloderma croceum. Using the Mimicking Intelligent Read Assembly (MIRA) and Trinity assembler, the OakContigDF159.1 hybrid assembly, containing 65 712 contigs with a mean length of 1003 bp, was constructed, giving broad coverage of metabolic pathways. This allowed us to identify 3018 oak contigs that were differentially expressed in EMs, with genes encoding proline-rich cell wall proteins and ethylene signalling-related transcription factors showing up-regulation while auxin and defence-related genes were down-regulated. In addition to the first report of remorin expression in EMs, the extensive coverage provided by the study permitted detection of differential regulation within large gene families (nitrogen, phosphorus and sugar transporters, aquaporins). This might indicate specific mechanisms of genome regulation in oak EMs compared with other trees. PMID:23672230
Tarkka, Mika T; Herrmann, Sylvie; Wubet, Tesfaye; Feldhahn, Lasse; Recht, Sabine; Kurth, Florence; Mailänder, Sarah; Bönn, Markus; Neef, Maren; Angay, Oguzhan; Bacht, Michael; Graf, Marcel; Maboreke, Hazel; Fleischmann, Frank; Grams, Thorsten E E; Ruess, Liliane; Schädler, Martin; Brandl, Roland; Scheu, Stefan; Schrey, Silvia D; Grosse, Ivo; Buscot, François
The propositional content of a reference is the proposition attributing to the referent theproperties that correspond to the nouns and modifiers in the reference (for example, thepropositional content of `Mary\\
Janyce M. Wiebe
National Technical Information Service (NTIS)
This paper addresses the most common system for providing aircraft heading reference: the magnetic heading reference system. It begins briefly explaining the importance of a magnetic heading reference. It then addresses the fundamental characteristics of ...
D. L. Moore
Joseph B. James and Fred J. Genthner United States Environmental Protection Agency, Gulf Breeze, FL Background: Methods using rapid cycle, real-time, quantitative (QPCR) are being developed for detecting and quantifying Enterococcus spp. as well as other aquatic b...
Development of a Reference Standard of Escherichia coli DNA for Residual DNA Determination in China.
This collaborative study developed the first national Escherichia coli (E. coli) DNA reference standard for standardizing quantitative residual DNA assay methods, fluorescence dye (PicoGreen) and quantitative PCR (q-PCR), which were widely employed to measure residual DNA contents of prokaryotic-derived recombinant products. High purity of E. coli strain BL21 was extracted by the cetyl triethyl ammonium bromide (CTAB)/phenol chloroform method, analyzed by UV-visible spectrophotometry and electrophoresis, diluted with tris-EDTA (TE) buffer and manually dispensed. Then, with a cooperative calibration among six laboratories, including five manufacturers and one national control laboratory, the concentration of E. coli DNA standard solution was determined as 96.2 ?g/mL (95% C.I: 95.5-96.9 ?g/mL, CV 3.4%). The candidate showed excellent stability both from accelerated degradation study and real time stability study. The applicability study showed that the E. coli DNA reference could reach the sensitivity of 0.781 ng/mL and 1 fg/?L, respectively, in fluorescent dye and q-PCR assay, and also had good linearity and precision. The consistency of the reference could meet the requirements of the national reference standard. As a conclusion, the candidate material was suitable to serve as a China national standard for E. coli residual DNA determination. The successful establishment of the E. coli DNA standard will facilitate the standardization of quantitative methods for testing residual host cell DNA. PMID:24086318
Wang, Lan; Rao, Chunming; Gao, Kai; Li, Yonghong; Fu, Zhihao; Bi, Hua; Wang, Junzhi
Development of a Reference Standard of Escherichia coli DNA for Residual DNA Determination in China
This collaborative study developed the first national Escherichia coli (E. coli) DNA reference standard for standardizing quantitative residual DNA assay methods, fluorescence dye (PicoGreen) and quantitative PCR (q-PCR), which were widely employed to measure residual DNA contents of prokaryotic-derived recombinant products. High purity of E. coli strain BL21 was extracted by the cetyl triethyl ammonium bromide (CTAB)/phenol chloroform method, analyzed by UV-visible spectrophotometry and electrophoresis, diluted with tris-EDTA (TE) buffer and manually dispensed. Then, with a cooperative calibration among six laboratories, including five manufacturers and one national control laboratory, the concentration of E. coli DNA standard solution was determined as 96.2 ?g/mL (95% C.I: 95.5–96.9 ?g/mL, CV 3.4%). The candidate showed excellent stability both from accelerated degradation study and real time stability study. The applicability study showed that the E. coli DNA reference could reach the sensitivity of 0.781 ng/mL and 1 fg/?L, respectively, in fluorescent dye and q-PCR assay, and also had good linearity and precision. The consistency of the reference could meet the requirements of the national reference standard. As a conclusion, the candidate material was suitable to serve as a China national standard for E. coli residual DNA determination. The successful establishment of the E. coli DNA standard will facilitate the standardization of quantitative methods for testing residual host cell DNA.
Gao, Kai; Li, Yonghong; Fu, Zhihao; Bi, Hua; Wang, Junzhi
Escherichia coli and Enterococcus spp. in rainwater tank samples: comparison of culture-based methods and 23S rRNA gene quantitative PCR assays.
In this study, culture-based methods and quantitative PCR (qPCR) assays were compared with each other for the measurement of Escherichia coli and Enterococcus spp. in water samples collected from rainwater tanks in Southeast Queensland, Australia. Among the 50 rainwater tank samples tested, 26 (52%) and 46 (92%) samples yielded E. coli numbers as measured by EPA Method 1603 and E. coli 23S rRNA gene qPCR assay, respectively. Similarly, 49 (98%) and 47 (94%) samples yielded Enterococcus spp. numbers as measured by EPA Method 1600 and Enterococcus spp. 23S rRNA gene qPCR assay, respectively. The mean E. coli (2.49 ± 0.85) log(10) and Enterococcus spp. (2.72 ± 0.32) log(10) numbers as measured by qPCR assays were significantly (P < 0001) different than E. coli (0.91 ± 0.80) log(10) and Enterococcus spp. (1.86 ± 0.60) log(10) numbers as measured by culture-based method. Weak but significant correlations were observed between both EPA Method 1603 and the E. coli qPCR assay (r = 0.47, P = 0.0009), and EPA Method 1600 and the Enterococcus spp. qPCR assay (r = 0.42, P = 0.002). Good qualitative agreement was found between the culture-based method and the Enterococcus spp. qPCR assay in terms of detecting fecal pollution in water samples from the studied rainwater tanks. More research studies, however, are needed to shed some light on the discrepancies associated with the culture-based methods and qPCR assays for measuring fecal indicator bacteria. PMID:22963205
Ahmed, W; Richardson, K; Sidhu, J P S; Toze, S
Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains
The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.
Ritalahti, Kirsti M.; Amos, Benjamin K.; Sung, Youlboong; Wu, Qingzhong; Koenigsberg, Stephen S.; Loffler, Frank E.
Age-related changes in the expression of schizophrenia susceptibility genes in the human prefrontal cortex
The molecular basis of complex neuropsychiatric disorders most likely involves many genes. In recent years, specific genetic\\u000a variations influencing risk for schizophrenia and other neuropsychiatric disorders have been reported. We have used custom\\u000a DNA microarrays and qPCR to investigate the expression of putative schizophrenia susceptibility genes and related genes of\\u000a interest in the normal human brain. Expression of 31 genes
Carlo Colantuoni; Thomas M. Hyde; Shruti Mitkus; Andrew Joseph; Leah Sartorius; Claudia Aguirre; Johanna Creswell; Elizabeth Johnson; Amy Deep-Soboslay; Mary M. Herman; Barbara K. Lipska; Daniel R. Weinberger; Joel E. Kleinman
Reach for Reference. Four Recent Reference Books
ERIC Educational Resources Information Center
This article provides descriptions of four new science and technology encyclopedias that are appropriate for inclusion in upper elementary and/or middle school reference collections. "The Macmillan Encyclopedia of Weather" (Stern, Macmillan Reference/Gale), a one-volume encyclopedia for upper elementary and middle level students, is a…
Safford, Barbara Ripp
Reach for Reference. Four Recent Reference Books
ERIC Educational Resources Information Center
|This article provides descriptions of four new science and technology encyclopedias that are appropriate for inclusion in upper elementary and/or middle school reference collections. "The Macmillan Encyclopedia of Weather" (Stern, Macmillan Reference/Gale), a one-volume encyclopedia for upper elementary and middle level students, is a…
Safford, Barbara Ripp
Verifying Reference Counted Objects
Reference counting is a pervasive resource management idiom where each resource contains a count of the number of clients that hold a reference to it, and each client increments (resp., de cre- ments) the count on acquiring (resp., releasing) the resour ce. If the reference count falls to zero, the system can reclaim the res ource. Reference counting is often
Michael Emmi; Ranjit Jhala; Rupak Majumdar
ERIC Educational Resources Information Center
|The all-in-one "Reference reference" you've been waiting for, this invaluable book offers a concise introduction to reference sources and services for a variety of readers, from library staff members who are asked to work in the reference department to managers and others who wish to familiarize themselves with this important area of…
Mulac, Carolyn M.
ERIC Educational Resources Information Center
|Discusses digital reference services, also known as virtual reference, chat reference, or online reference, based on a round table discussion at the 2002 American Library Association annual conference in Atlanta. Topics include numbers and marketing; sustainability; competition and models; evaluation methods; outsourcing; staffing and training;…
Kenney, Brian
Gene manipulated peritoneal cell patch repairs infarcted myocardium
A gene manipulated cell patch using a homologous peritoneum substrate was developed and applied after myocardial infarction to repair scarred myocardium. We genetically engineered male rat mesenchymal stem cells (MSC) using adenoviral transduction to over-express CXCR4\\/green fluorescent protein (GFP) (MSCCXCR4) or MSCNull or siRNA targeting CXCR4 (MSCsiRNA). Gene expression was studied by real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay
Wei Huang; Dongsheng Zhang; Ronald W. Millard; Tao Wang; Tiemin Zhao; Guo-Chang Fan; Atif Ashraf; Meifeng Xu; Muhammad Ashraf; Yigang Wang
Quantitative multi-gene expression profiling of primary prostate cancer
BACKGROUND. This study describes the evaluation of the expression patterns of prostate- related transcripts in 106 matched prostate tissues from prostatectomies as predictors for prostate cancer (PCa). METHODS. Quantitative PCR (QPCR) assays with site-specific hybridization probes were established for four housekeeping genes (GAPDH, HPRT, PBGD, TBP) and nine prostate- related genes (AibZIP, D-GPCR, EZH2, PCA3, PDEF, prostein, PSA, PSCA, TRPM8).
Uta Schmidt; Susanne Fuessel; Rainer Koch; Gustavo B. Baretton; Andrea Lohse; Silke Tomasetti; Susanne Unversucht; Michael Froehner; Manfred P. Wirth; Axel Meye
ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs
Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.
Longo, Ana V.; Rodriguez, David; da Silva Leite, Domingos; Toledo, Luis Felipe; Mendoza Almeralla, Cinthya; Burrowes, Patricia A.; Zamudio, Kelly R.
Assessment of microzooplankton grazing on Heterosigma akashiwo using a species-specific approach combining quantitative real-time PCR (QPCR) and dilution methods.
Delaware's Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top-down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species-specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific microzooplankton grazing pressure on H akashiwo ranged from 0.88 to 1.88 day(-1) at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplankton species within a mixed community. PMID:17609846
Demir, Elif; Coyne, Kathryn J; Doblin, Martina A; Handy, Sara M; Hutchins, David A
ITS1 copy number varies among Batrachochytrium dendrobatidis strains: implications for qPCR estimates of infection intensity from field-collected amphibian skin swabs.
Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians. PMID:23555682
Longo, Ana V; Rodriguez, David; da Silva Leite, Domingos; Toledo, Luís Felipe; Mendoza Almeralla, Cinthya; Burrowes, Patricia A; Zamudio, Kelly R
Accurate Optical Reference Catalogs.
National Technical Information Service (NTIS)
This paper reviews current and near future all-sky astrometric catalogs on the International Celestial Reference Frame (ICRF) with an emphasis on reference star data at optical wavelengths for user applications.
N. Zacharias
Code of Federal Regulations, 2010 CFR
...2010-07-01 false References: Cross Pensions, Bonuses, and Veterans...service requires that the initial infection must have occurred during active...Authority: 38 U.S.C. 105(a)) Cross-References: In line of duty....
Verifying Reference Counting Implementations
Reference counting is a widely-used resource management idiom which maintains a count of references to each resource by incrementing the count upon an acquisition, and decrementing upon a release; resources whose counts fall to zero may be recycled. We present an algorithm to verify the correctness of reference counting with minimal user interaction. Our algorithm performs com- positional verification through
Michael Emmi; Ranjit Jhala; Eddie Kohler; Rupak Majumdar
Academic Library Reference Services.
ERIC Educational Resources Information Center
|This examination of the philosophy and objectives of academic library reference services provides an overview of the major reference approaches to fulfilling the following primary objectives of reference services: (1) providing accurate answers to patrons' questions and/or helping patrons find sources to pursue their research needs; (2) building…
Batt, Fred
The delivery of library reference service can be practically supplemented through the appropriate incorporation and use of software tools commonly referred to as expert systems. The level of support such systems can afford the reference service organization is dependent on the degree of complexity characteristic of the rule-based programming techniques used to develop a particular system and the size of
Dana E. Smith
Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR
BACKGROUND: Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16
Ian Roberts; Grace Ng; Nicola Foster; Margaret Stanley; Michael T Herdman; Mark R Pett; Andrew Teschendorff; Nicholas Coleman
Spectrum and Prevalence of Mutations Involving BrS1-12-Susceptibility Genes in a Cohort of Unrelated Patients Referred for Brugada Syndrome Genetic Testing: Implications for Genetic Testing
Objective To provide the spectrum and prevalence of mutations in the 12 Brugada Syndrome (BrS)-susceptibility genes discovered to date, in a single large BrS cohort. Background BrS is a potentially lethal heritable arrhythmia syndrome diagnosed electrocardiographically by coved-type ST segment elevation in the right precordial leads (V1-V3; type-1 Brugada ECG pattern) and the presence of a personal/family history of cardiac events. Methods Using PCR, DHPLC, and DNA sequencing, comprehensive mutational analysis of BrS1-12-susceptibility genes was performed in 129 unrelated patients with possible/probable BrS [46 with clinically diagnosed BrS (ECG pattern plus personal/family history of a cardiac event) and 83 with type 1 ECG pattern only]. Results Overall, 27 (21%) patients had a putative pathogenic mutation, absent in 1400 Caucasian reference alleles, including 21 patients with an SCN5A mutation, 2 CACNB2B, 1 KCNJ8, 1 KCND3, 1 SCN1Bb, and 1 HCN4. The overall mutation yield was 23% in type 1 ECG pattern only patients versus 17% in clinically diagnosed BrS patients, was significantly greater among young men < 20 years of age with clinically diagnosed BrS, and among patients who had a prolonged PQ interval. Conclusions We identified putative pathogenic mutations in ~20% of our BrS cohort, with BrS2-12 accounting for < 5%. Importantly, the yield was similar between patients with only a type 1 BrS ECG pattern and those with clinically established BrS. The yield approaches 40% for SCN5A-mediated BrS (BrS1) when the PQ interval exceeds 200ms. Calcium channel-mediated BrS is extremely unlikely in the absence of a short QT interval.
Crotti, Lia; Kellen, Cherisse A.; Tester, David J.; Castelletti, Silvia; Giudicessi, John R.; Torchio, Margherita; Medeiros-Domingo, Argelia; Simone, Savastano; Will, Melissa L; Dagradi, Federica; Schwartz, Peter J.; Ackerman, Michael J.
High frequency reference electrode
A high frequency reference electrode for electrochemical experiments comprises a mercury-calomel or silver-silver chloride reference electrode with a layer of platinum around it and a layer of a chemically and electrically resistant material such as TEFLON around the platinum covering all but a small ring or halo' at the tip of the reference electrode, adjacent to the active portion of the reference electrode. The voltage output of the platinum layer, which serves as a redox electrode, and that of the reference electrode are coupled by a capacitor or a set of capacitors and the coupled output transmitted to a standard laboratory potentiostat. The platinum may be applied by thermal decomposition to the surface of the reference electrode. The electrode provides superior high-frequency response over conventional electrodes. 4 figs.
Kronberg, J.W.
High frequency reference electrode
A high frequency reference electrode for electrochemical experiments comprises a mercury-calomel or silver-silver chloride reference electrode with a layer of platinum around it and a layer of a chemically and electrically resistant material such as TEFLON around the platinum covering all but a small ring or halo'' at the tip of the reference electrode, adjacent to the active portion of the reference electrode. The voltage output of the platinum layer, which serves as a redox electrode, and that of the reference electrode are coupled by a capacitor or a set of capacitors and the coupled output transmitted to a standard laboratory potentiostat. The platinum may be applied by thermal decomposition to the surface of the reference electrode. The electrode provides superior high-frequency response over conventional electrodes.
Kronberg, J.W.
Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis.
This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection. PMID:21075527
Ramos, Carlos A N; Araújo, Flábio R; Souza, Ingrid I F; Bacanelli, G; Luiz, Hera L; Russi, Lívia S; Oliveira, Renato H M; Soares, Cleber O; Rosinha, Grácia M S; Alves, Leucio C
Rapid detection of the Vibrio cholerae ctx gene in food enrichments using real-time polymerase chain reaction.
A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42 degrees C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35 degrees C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42OC for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98% (88/90) and 100% (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87% (78/90) and 83% (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 1-2 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products. PMID:17955973
Fedio, Willis; Blackstone, George M; Kikuta-Oshima, Lynne; Wendakoon, Chitra; McGrath, Timothy H; DePaola, Angelo
An optical voltage reference for providing an alternative to a battery source is described. The optical reference apparatus provides a temperature stable, high precision, isolated voltage reference through the use of optical isolation techniques to eliminate current and impedance coupling errors. Pulse rate frequency modulation is employed to eliminate errors in the optical transmission link while phase-lock feedback is employed to stabilize the frequency to voltage transfer function. 2 figures.
Rankin, R.; Kotter, D.
An optical voltage reference for providing an alternative to a battery source. The optical reference apparatus provides a temperature stable, high precision, isolated voltage reference through the use of optical isolation techniques to eliminate current and impedance coupling errors. Pulse rate frequency modulation is employed to eliminate errors in the optical transmission link while phase-lock feedback is employed to stabilize the frequency to voltage transfer function.
Rankin, R.; Kotter, D.
Genetics Home Reference: Retinoblastoma
... type of eye cancer that usually develops in early childhood, typically before the age of 5. This form ... copies of the RB1 gene. Then, usually in early childhood, both copies of the RB1 gene in retinal ...
Additional Reference Information
Center for Biologics Evaluation and Research (CBER)
... Additional Reference Article Information. Grace Chai, Laura Governale, Patty Greene, Hina Mehta, Vicky Borders-Hemphill, David Money. ... More results from www.fda.gov/advisorycommittees/committeesmeetingmaterials/pediatricadvisorycommittee
Martindale's "The Reference Desk"
NSDL National Science Digital Library
This online reference provides links to reference materials on a variety of science topics, including astronomy, biosciences, chemistry, engineering, geosciences, and many others. The links are organized by topics (education, entertainment, music, science, and so forth). Within each topic, the resources are arranged by type (dictionaries, databases, journals, web sites, and so forth).
Martindale, Jim
Reference Service Training Manual.
ERIC Educational Resources Information Center
|This manual is primarily designed as an orientation program for librarians new to the Reference Services Division at the State University of New York at Albany University Libraries. It contains practical information and some of the procedures necessary for providing service at the reference desk in these libraries. The appendices provide samples…
Mielke, John; Young, William
ERIC Educational Resources Information Center
To help librarians answer patrons' questions about art works, especially paintings, the procedures followed by the reference division of the Suffolk Cooperative Library System are outlined, and a list of reference materials is suggested. Topics covered include biographical information about artists, identification of paintings, location of…
Lukac, Milan
Reference Collection Development Policy.
ERIC Educational Resources Information Center
The purpose of this policy is to outline those principles that guide the development of the Reference Collection at the Calvin T. Ryan Library, University of Nebraska at Kearney. It clarifies, records, and makes public the basic principles that the members of the Reference Department believe should be applied in adding items to or withdrawing…
Nebraska Univ., Kearney.
ERIC Educational Resources Information Center
Elementary students are introduced to a wide variety of reference materials. Separate sections contain activities for using an atlas, "The Book of Lists," dictionaries, encyclopedias, "Familiar Quotations," games, "The Guiness Book of World Records," magazines, multi-references, newspapers, the "T.V. Guide," and world maps. Students complete…
Bolger, Charlene
ERIC Educational Resources Information Center
|Relates the marketing concept to library reference services. Highlights include a review of the literature and an overview of marketing, including research, the marketing mix, strategic plan, marketing plan, and marketing audit. Marketing principles are applied to reference services through the marketing mix elements of product, price, place, and…
Norman, O. Gene
Leading Education Reference Sources.
ERIC Educational Resources Information Center
|This annotated bibliography focuses on, but is not limited to, reference sources on education found in the library at St. Bonaventure University, New York. The ERIC database leads the list of leading education reference sources. Also mentioned are the "Thesaurus of ERIC Descriptors" and the Academic Index (InfoTrak) computer system. Other…
Spencer, Michael D.
Reference Service Training Manual.
ERIC Educational Resources Information Center
This manual is primarily designed as an orientation program for librarians new to the Reference Services Division at the State University of New York at Albany University Libraries. It contains practical information and some of the procedures necessary for providing service at the reference desk in these libraries. The appendices provide samples…
Mielke, John; Young, William
ERIC Educational Resources Information Center
|Good reference librarians need to be good interviewers. Unfortunately most librarians have not learned to use the technique of open questions to obtain information about the user and what he is looking for. Examples of open and closed questions with typical responses are given. (4 references) (DH)|
King, Geraldine B.
Purpose – The purpose of this paper is to discuss whether the outcomes of a reference transaction can be improved by focusing on the ethical dimension of the conversation between user and librarian. The aim is to provide a better understanding of the ethical implications of the reference interview. Design\\/methodology\\/approach – The starting point is the ethical questions raised by
Synnřve Ulvik; Gunhild Salvesen
Biogenic amines content in Spanish and French natural ciders: application of qPCR for quantitative detection of biogenic amine-producers.
Biogenic amines (BA) are low molecular weight nitrogenous bases commonly found in fermented foods and beverages and their consumption can induce undesirable reactions. In this work, the BA content in natural cider from Spain and France was determined. Samples from commercially available cider or obtained during the elaboration process were analyzed. A different profile and BA concentration was observed depending on cider origin. qPCR tools developed for the quantitative detection of BA producers from cheese and wine were tested in the cider samples. A good connection between the BA content and the presence of BA-producing microorganisms was observed. Based on these tools, BA-producing bacteria were isolated from the analyzed cider samples, including new potential histamine- and putrescine-producing Lactobacillus paracollinoides strains. PMID:21356464
Ladero, Victor; Coton, Monika; Fernández, María; Buron, Nicolas; Martín, M Cruz; Guichard, Hugues; Coton, Emmanuel; Alvarez, Miguel A
Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles
BACKGROUND: Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in
Ken-Go Hayashi; Koichi Ushizawa; Misa Hosoe; Toru Takahashi
An examination of the utility of a nuclear DNA/mitochondrial DNA duplex qPCR assay to assess surface decontamination of hair.
The goal of this study was to compare two commonly used methods for the surface decontamination of human hair shafts, and to evaluate the use of a duplex real-time qPCR assay to assess decontamination effectiveness for the purpose of mitochondrial DNA typing. Hair shafts of known mitochondrial DNA haplotype were coated with undiluted saliva, semen or blood, each of known mitochondrial haplotype distinct from the test hair. Surface decontamination was conducted by enzymatic treatment with Terg-a-zyme™ and by chemical treatment with dilutions of sodium hypochlorite (NaClO, bleach). Following DNA extraction, a duplex (nuclear and mitochondrial DNA) real-time qPCR assay was used to quantify mitochondrial DNA and to test for surface contamination by quantifying the exogenous nuclear DNA not removed from the hair shaft. The NaClO treatment was found to be more effective for removing surface contamination than the Terg-a-zyme™ treatment, and it was procedurally simpler to implement, resulting in a significant savings of sample processing time. Exposure to 3% NaClO for up to two minutes had no detrimental effect on quantity or typing of the mitochondrial DNA belonging to the hair. In addition, we demonstrated that the duplex real-time PCR assay is a convenient early-warning diagnostic method for the detection of the presence of external DNA contamination, providing an assessment of the purity of the sample prior to embarking on further analysis by more laborious mitochondrial DNA typing methods. PMID:23582697
Date-Chong, Mavis; Buoncristiani, Martin R; Aceves, Margaret; Orrego, Cristián
Enumeration of bacteriophage particles: Comparative analysis of the traditional plaque assay and real-time QPCR- and nanosight-based assays.
Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current "gold standard" for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations. PMID:22334864
Anderson, Bradley; Rashid, Mohammed H; Carter, Chandi; Pasternack, Gary; Rajanna, Chythanya; Revazishvili, Tamara; Dean, Timothy; Senecal, Andre; Sulakvelidze, Alexander
Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.
Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...
Gene expression underlying adaptive variation in Heliconius wing patterns: non-modular regulation of overlapping cinnabar and vermilion prepatterns
Geographical variation in the mimetic wing patterns of the butterfly Heliconius erato is a textbook example of adaptive polymorphism; however, little is known about how this variation is controlled developmentally. Using microarrays and qPCR, we identified and compared expression of candidate genes potentially involved with a red\\/yellow forewing band polymorphism in H. erato. We found that transcripts encoding the pigment
Robert D. Reed; W. Owen McMillan; Lisa M. Nagy
Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains
The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA,
Kirsti M. Ritalahti; Benjamin K. Amos; Youlboong Sung; Qingzhong Wu; Stephen S. Koenigsberg; Frank E. Loffler
The subduction reference framework
NASA Astrophysics Data System (ADS)
Plate tectonic reconstructions are essential for determining the spatial and temporal context for geological and geophysical data and help distinguish competing models for regional plate kinematic histories and the relationships between tectonic features and events. Plate reconstructions, a series of relative plate motions anchored to an absolute reference frame via a plate circuit, can act as surface boundary constraints for mantle convection models, allowing us to link surface processes to the deep earth. One of the limitations in global plate motion models has been to accurately determine the positions of plates through time. Traditionally, this has been based on either palaeomagnetic or hotspot reference frames, however both these methodologies have some shortcomings. Palaeomagnetic reference frames can determine latitudes but not longitudes, with additional inaccuracies due to true polar wander. Hotspot reference frames can only be confidently tied back to about 130 Ma and there is evidence that mantle plumes have moved relative to each other. New "hybrid" reference frames are emerging, which consist of fixed or moving hotspot reference frames merged with true polar wander (TPW) corrected palaeomagnetic reference frames. We have devised a methodology to link plate reconstructions to mantle convection back to Pangaea breakup time to converge on a solution that correctly aligns slab material in the mantle to the locations of subduction zones in the past. We aim to construct a "Subduction Reference Frame" for plate motions since 200 Ma by iteratively matching forward geodynamic models with tomographically imaged slabs in the mantle. Our forward models involve coupling global plate kinematics, the thermal structure of the oceanic lithosphere and slab assimilation to a spherical mantle convection code, CitcomS. Preliminary results have been obtained for a plate motion model using a moving hotspot reference frame to 100 Ma and a TPW corrected reference frame for times prior to 100 Ma. Focussing on the Farallon slab and the palaeo-subduction east of Australia, we find that our models reasonably reproduce the present-day location of the Farallon slab. However, there is a mismatch between the slab east of Australia and the predicted location of subduction based on the TPW-corrected reference frame. Further models will allow us to test new alternative reference frames to achieve a correct alignment with the location of slabs imaged in the mantle and the location of subduction along continental margins in the past.
Seton, M.; Müller, D.; Gurnis, M.; Flament, N.; Whittaker, J.
NSDL National Science Digital Library
This interactive resourece is from the Museum's Seminars on Science, a series of distance-learning courses designed to help educators meet the new national science standards. Part of the Frontiers in Physical Science seminar, the Frames of Reference interactive has three sections. An animated overview illustrates why whether an object appears to be at rest or in motion depends on your frame of reference, a nine-question interactive test that allows you to check your understanding of the concept and there is a matrix that allows you to view any of the nine frame-of-reference combinations. Shockwave is required to view the site.
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