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Patent application title: Method for high-throughput gene expression profile analysis
Honghua Li (Monmouth Junction, NJ, US)
Guohong Hu (Piscataway, NJ, US)
Qifeng Yang (Highland Park, NJ, US)
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2010-11-25
Patent application number: 20100297622
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Patent application title: Method for high-throughput gene expression profile analysis
Guohong Hu
Honghua Li
Qifeng Yang
Jane Massey Licata;Licata & Tyrrell P.C.
Origin: MARLTON, NJ US
IPC8 Class: AC12Q168FI
USPC Class:
Publication date: 11/25/2010
Patent application number: 20100297622
The present invention embraces a method for high-throughput gene profiling
with high specificity and sensitivity. With this system, >1000 mRNA
species can be co-amplified using gene-specific primers from a single
cell. The primers are designed to amplify sequences of desirable length,
which are in different exons. The exons can be either adjacent and
separated by a large intron or include more than two exons. The amplified
sequences are then analyzed by microarray with probes hybridizing to
neighboring exons.
1. A method for high-throughput gene expression profile analysis
comprising(I) synthesizing a plurality of primers and probes for a set of
genes of interest by:(a) determining the intron-exon structure of each
gene of interest;(b) selecting for each gene of interest a probe, wherein
said probe:(i) is less than 50 bases in length, and(ii) anneals to two
adjacent exons flanking an intron of at least 200 bases in length;(c)
subsequently selecting for each gene of interest a pair of primers,
wherein said primers:(i) are less than 50 bases in length,(ii) anneal to
two exons of the gene of interest,(v) produce an amplicon of less than
200 base pairs in length, and(vi) when compared to sequences in one or
more databases of genomic or mRNA sequences, fail to amplify a
non-specific sequence in silico; and(d) subsequently selecting a nested
primer, wherein said nested primer:(i) anneals to the amplicon of step
(c)(ii), and(ii) produces, in an amplification reaction, a
single-stranded nucleic acid molecule hybridizable with the probe of step
(b); and(II) subjecting a sample to an amplification reaction with the
plurality of primers of step (I)(c), thereby producing double-stranded
amplicons;(III) amplifying single-stranded nucleic acid molecules from
the double-stranded amplicons with the plurality of nested primers of
step (I)(d); and(IV) quantifying the single-stranded nucleic molecules
via hybridization with the plurality of probes of step (I)(b) thereby
determining the expression profile of the set of genes of interest in the
2. The method of claim 1, wherein said probe has a melting temperature of between 50.degree. C. and 60.degree. C., has a GC content of between 35% and 70%, complements less than nine contiguous bases or 11 contiguous bases with a gap of any primer or probe of the plurality of primers and probes, and complements at least 14 contiguous bases or 18 contiguous bases with a gap of the gene of interest.
3. The method of claim 1, wherein the primers of said pair of primers have a melting temperature of between 46.degree. C. and 56.degree. C., have a GC content of between 35% and 70%, complement less than nine contiguous bases or 12 contiguous bases with a gap of any other primer of the plurality of primers, and complement at least 14 contiguous bases or 18 contiguous bases with a gap of the gene of interest.
4. The method of claim 1, wherein the probe is labeled.
[0002]Biological processes are underlain by interactions between various genes, their products, and defined pathways in the molecular networks. Global gene expression profiling of cells and tissues under physiological or in vitro conditions facilitates the understanding of the correlation between gene function and phenotypic effects. The advent of the microarray-based high-throughput RNA detection system (Schena, et al. (1995) Science 270:467-470; Lockhart, et al. (1996) Nat. Biotechnol. 14(13):1675-1680) has made it possible to profile gene expression patterns for the entire transcriptome. However, to specifically detect individual transcripts, discrimination between closely related sequences, including the genomic sequence, pseudogenes, and unprocessed RNA, is essential. Although contamination with genomic DNA may not be a concern for applications using purified mRNA, gene sequences must be taken into consideration for applications using cell lysate directly without RNA extraction. This becomes especially important when the transcripts under study are present at low abundance. As indicated, pseudogenes and their possible transcripts are also of concern. The number of pseudogenes in the human genome has been estimated to be 20,000 to 33,000 (Goncalves, et al. (2000) Genome Res. 10(5):672-678; Harrison, et al. (2002) Genome Res. 12(2):272-280), and usually share a high degree of sequence identity with the closely related genes.
[0003]Among the microarray-based platforms, GENECHIP (AFFYMETRIX, Santa Clara, Calif.) is a commonly used system that has contributed to the understanding of complex gene expression networks. However, because this technology is limited by its high degree of nonspecificity and insensitivity, its application has been limited in molecular network integration. Analysis has indicated that 20,696 (10.5%) probes on the GENECHIP U95A/Av2 Array are nonspecific and 18,363 (9.3%) probes missed the target transcript sequences (Zhang, et al. (2005) Genomics 85(3):297-308). The numbers of nonspecific and mis-targeted probes on the U133A array are comparable at 29,405 (12.1%) and 19,717 (8.0%), respectively (Zhang, et al. (2005) supra). These problematic probes certainly and substantially compromise data accuracy, decrease the value of microarray data, and are not acceptable in the study of molecular network integration.
[0004]High-throughput gene expression profiling with superior sensitivity is becoming more and more attractive. For example, in breast cancer research, analysis of specimens from microdissection can provide important information about genes involved in different cancer development stages. In most applications, gene expression profiling with microarrays, including GENECHIP, requires amplification of sample RNA. Conventionally, 1 to 3 μg of RNA is required for each assay. However, specimens from fine needle biopsy typically only contain a limited number of cells. The ability to analyze a large number of genes in single cells would facilitate our understanding of the origin and clonality of cancer development and provide the molecular details involved in different stages of the cell cycle.
[0005]Current methodologies for gene expression profiling in small RNA samples, especially those from single cells, are very limited. Many of these protocols require multiple enzymatic reactions that may seriously reduce the sensitivity and compromise the specificity. RNA preparation in most of applications also involves a number of steps, which is rather lengthy, tedious, and requires highly skilled personnel.
[0006]A number of strategies have been used to enhance the multiplex amplification capacity. For example, optimization of polymerase chain reaction (PCR) conditions has been used to enhance multiplex amplification capacity. While optimizing PCR conditions may allow more sequences to be amplified simultaneously, the major factor limiting amplification of multiple amplicons is primer-primer interaction and optimized PCR conditions cannot reduce the amount of interaction between primers. For example, when n PCR primer pairs are combined in one reaction, the possible interacting pairs between these primers would be 2n2+n, many of which may result in a nonspecific product.
[0007]Another strategy for increasing multiplexing capacity involves the use of universal sequences at the 5'-ends of all specific primers. Attaching these sequences during the early stage of amplification enables multiplex amplification of 26 to >1000 sequences in a single reaction. However, since attaching universal sequences involves multiple enzymatic reactions and does not eliminate primer-primer interactions, amplification capacity and sensitivity are still limited with this approach.
[0008]Thus there remains a need for improved gene expression profiling.
[0009]The present invention embraces a method for high-throughput gene expression profile analysis. More specifically, the invention relates to the design of primers and probes for large-scale gene expression profiling, which provide high specificity and sensitivity. The present method involves the steps of:
[0010](I) synthesizing a plurality of primers and probes for a set of genes of interest by: [0011](a) determining the intron-exon structure of each gene of interest; [0012](b) selecting for each gene of interest a probe, wherein said probe: [0013](i) is less than 50 bases in length, and [0014](ii) anneals to two adjacent exons flanking an intron of at least 200 bases in length; [0015](c) subsequently selecting for each gene of interest a pair of primers, wherein said primers: [0016](i) are less than 50 bases in length, [0017](ii) anneal to two exons of the gene of interest, [0018](iii) produce an amplicon of less than 200 base pairs in length, and [0019](iv) when compared to sequences in one or more databases of genomic or mRNA sequences, fail to amplify a non-specific sequence in silicon and [0020](d) subsequently selecting a nested primer, wherein said nested primer: [0021](i) anneals to the amplicon of step (c)(ii), and [0022](ii) produces, in an amplification reaction, a single-stranded nucleic acid molecule hybridizable with the probe of step (b); and
[0023](II) subjecting a sample to an amplification reaction with the plurality of primers of step (I)(c), thereby producing double-stranded amplicons;
[0024](III) amplifying single-stranded nucleic acid molecules from the double-stranded amplicons with the plurality of nested primers of step (I)(d); and
[0025](IV) quantifying the single-stranded nucleic molecules via hybridization with the plurality of probes of step (I)(b) thereby determining the expression profile of the set of genes of interest in the sample.
[0026]According to certain embodiments, the probe of the instant method has a melting temperature of between 50° C. and 60° C., has a GC content of between 35% and 70%, complements less than nine contiguous bases or 11 contiguous bases with a gap of any primer or probe of the plurality of primers and probes, and complements at least 14 contiguous bases or 18 contiguous bases with a gap of the gene of interest. In particular embodiments, the probe is labeled.
[0027]According to other embodiments, the primers of said pair of primers have a melting temperature of between 46° C. and 56° C., have a GC content of between 35% and 70%, complement less than nine contiguous bases or 12 contiguous bases with a gap of any other primer of the plurality of primers, and complement at least 14 contiguous bases or 18 contiguous bases with a gap of the gene of interest.
[0028]Using the method of the invention, primers are specially designed to amplify processed RNA sequences very specifically. Probes used for microarray detection are designed only to hybridize to sequences amplified from processed RNA. A large number of RNA species directly released from very few cells or even single cells can be amplified to a detectable amount without RNA isolation. Amplified products can then be detected by the single-base extension assay on an oligonucleotide microarray.
[0029]FIG. 1A is a schematic illustration of the high-throughput gene expression profiling procedure.
[0030]FIG. 1B is a schematic illustration showing the selectivity of the primers and probes of the invention for processed RNA over genomic DNA, pseudogenes, and unprocessed RNA.
[0031]The present invention embraces a method for high-throughput gene expression profile analysis which involves designing primers and probes exhibiting a high degree of specificity and sensitivity. Compared with conventional gene expression profiling methods, the method of the invention is simple, safe, and very flexible and provides high specificity and high sensitivity.
[0032]As illustrated in FIG. 1A, the present invention involves the steps of synthesizing a plurality of primers and mRNA-specific probes for a set of genes of interest; subjecting a sample to an amplification reaction with the plurality of primers, thereby producing double-stranded amplicons; amplifying single-stranded nucleic acid molecules from the double-stranded amplicons with a plurality of nested primers; and quantifying the single-stranded nucleic molecules via hybridization with the plurality of probes thereby determining the expression profile of the set of genes of interest in the sample.
[0033]The method of the present invention is not limited by the nature of the gene of interest. In this regard, the gene of interest can be any gene, the expression product of which is sought to be detected in a sample. In this regard, a set of genes of interest can include genes encoding proteins or RNA involved in a particular pathological condition, responses to therapeutics, signal transduction, cell proliferation, responses to inflammatory stimuli, and the like. A "set of genes of interest" is intended as two or more genes. A set can be 10 genes, 50 genes, 100 genes or as many as 10000 genes, the expression product of which is sought to be detected in a sample. In accordance with particular embodiments of the present invention, an expression product is an RNA, e.g., a messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), or microRNA (miRNA).
[0034]In so far as 10, 50, 100 or 10000 or more genes or interest are analyzed according to the method of the invention, a "plurality of primers and probes" is commensurate with the number of genes of interest.
[0035]The specificity and sensitivity of the instant method is derived from the selection of the primers and probes used in the gene expression profiling. As shown in FIG. 1B, the primers and probes are designed so that they are specific for processed RNA, or amplification products thereof, and not unprocessed RNA, pseudogenes or genomic DNA. Accordingly, the plurality of primers and probes for a set of genes of interest are designed by first determining the location or structure of the introns and exons of each gene of interest. This step of the instant method can be carried out using experimental data and/or any conventional algorithm for determining intron-exon boundaries of a gene (e.g., GENSCAN or GENESPLICER). Such algorithms search the genomic sequence for typical splice sites, e.g., introns end with the dinucleotide ApG (3' splice site/acceptor) and start with the dinucleotide GpU (5' splice site/donor). By way of illustration, intron-exon boundaries can be identified by retrieving the genomic sequence of a gene of interest from a database (e.g., the UCSC genome database or NCBI GENBANK database) and comparing the genomic sequence to the mRNA sequence coding for the same protein, which can also be obtained from publicly assessable databases. Such comparisons can be carried out using readily available programs such as BLAT (Kent (2002) Genome Res. 12: 656-664), DIALIGN, CLUSTALW and the like. Stretches of sequences found in the genomic sequences, but not in the mRNA, are indicative of intronic sequences. Exon sequences are then used for primer and probe design, whereas intron sequences are useful for checking for possible interactions during the selection process of primers and probes. The promoter and downstream sequences of each gene can also be retrieved at the users' discretion for sequence comparisons.
[0036]Once the exon-intron structure of each gene of interest has been determined, probes specific for spanning exon boundaries (i.e., location where adjacent exons adjoin after the intron has been spliced) are selected. As used herein a "probe" is defined as an oligonucleotide or polynucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, an oligonucleotide or polynucleotide probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in oligonucleotide or polynucleotide probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, oligonucleotide probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. Oligonucleotide or polynucleotide probes may also be generically referred to as nucleic acid probes.
[0037]In particular embodiments, the probes of the present invention are less than 50 bases or nucleotides in length. In certain embodiments the probes are 15 to 50 bases in length, 20 to 40 bases in length, or more specifically 22 to 31 bases in length. As illustrated in FIG. 1B, the probes of the invention are specific for processed RNA, or amplification products thereof, because they anneal to two adjacent exons. In this regard, the probe is a "spanning probe" that anneals to separate (non-contiguous) portions of the gene of interest.
[0038]The terms "annealing" and "hybridization" are used interchangeably herein and mean the base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure. In certain embodiments, the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
[0039]In addition to annealing to two adjacent exons, particular embodiments of the present invention embrace the following criteria be used for probe selection: [0040]a. The probe matches its 3' exon not less than 2+a bp, but not more than 15-b bp, where "a" is the length of the nucleotide sequence shared by the intron and the 3' exon, and "b" is the length of the nucleotide sequence shared by the intron and the 5' exon; [0041]b. The probe matches any oligonucleotide in the plurality of primers or probes not more than 9 bp without gap, or 11 bp with a gap at the 3' end. In other words, the 3' end of the probe complements less than nine contiguous bases or 11 contiguous bases with a gap of any primer or probe of the plurality of primers and probes; [0042]c. The probe fails to anneal to any amplicon but its own annealing sequence in the set less than 14 bp without gap, or 18 bp with a gap, at its 3' end. Alternatively stated, the 3' end of the probe complements at least 14 contiguous bases or 18 contiguous bases with a gap of the gene of interest; [0043]d. The melting temperatures (Tm) of the probe is between 50 and 60° C.; and [0044]e. The GC content (GC %) of the probe is between 35% and 70%.
[0045]To ensure specificity of the probe for processed RNA, desirably the probe is selected to anneal to exons flanking an intron that is longer than an effectively amplifiable length, e.g., 200, 300 or 400 bases. For each gene of interest, it is desirable that the exons flanking the longest intron are selected for designing primers and probe to achieve the maximal amplification specificity. If no sequences meet the various criteria herein (i.e., GC %, Tm, complementarity with target and non-target sequences, etc.), exons flanking the next longest intron can be employed.
[0046]To facilitate detection and quantification, particular embodiments of the present invention embrace a labeled probe. The term "labeled probe" refers to a probe that provides a detectable signal. The invention is not limited by the nature of the label chosen. Therefore the label can include, but is not limited to, labels which include a dye or a radionucleotide (e.g., 32P), fluorescein moiety, a biotin moiety, luminogenic, fluorogenic, phosphorescent, or fluors in combination with moieties that can suppress emission by fluorescence energy transfer (FET). Numerous methods are available for the detection of nucleic acids containing any of the above-listed labels. For example, biotin-labeled probes may be detected using non-isotopic detection methods which employ streptavidin-alkaline phosphatase conjugates. Fluorescein-labeled probes may be detected using a fluorescein-imager. Further the probe may contain positively charged adducts (e.g., the Cy3 and Cy5 dyes) and/or positively charged amino acids to permit detection. In particular embodiments, the probe is selected to be next to a "C," or other appropriate base, at its 3' end for the purpose of labeling.
[0047]At each junction or boundary of the exons in the RNA, every possible probe sequence is analyzed according to above requirements and criteria, and the selected probe sequence should have the optimal Tm (closed to the median of allowed Tm range) to achieve the maximal Tm uniformity among the plurality of probes.
[0048]Once the optimal probe is selected, primers are subsequently selected. The term "primer" refers to a oligonucleotide that anneals to a template nucleic acid sequence and allows synthesis of a sequence complementary to the template nucleic acid. A "pair of primers" refers to primers in a set of at least two primers that are capable of exponentially amplifying a target nucleic acid in the polymerase chain reaction. In particular embodiments, the primers of the present invention are less than 50 bases or nucleotides in length. In certain embodiments the primers are 15 to 50 bases in length, 15 to 40 bases in length, or more specifically 20 to 25 bases in length. As illustrated in FIG. 1B, the primers of the invention are specific for processed RNA, or amplification products thereof, and not unprocessed RNA, pseudogenes or genomic DNA because each primer anneals to a different exon of the gene of interest and is capable of producing an amplifiable product. In accordance with primer selection, the pair of primers can anneal to two adjacent exons or two exons that are close enough to produce an amplifiable product.
[0049]The term "amplification product" or "amplicon" as used herein refers to the product of an amplification reaction. Exemplary amplification reactions include, but are not limited to, primer extension, the polymerase chain reaction, and the like. Thus, exemplary amplification products include, but are not limited to, primer extension products, PCR amplicons, and the like. A product is said to be an "amplifiable product" if it is of an appropriate length to be amplified. In accordance with the present invention, an amplifiable product is an amplicon of less than 200 base pairs in length, or more desirably less than 150 base pairs in length. In particular embodiments, the amplicon is 50 to 200 base pairs in length or more desirably, 70 to 150 base pairs in length.
[0050]In addition to annealing to two different exons, particular embodiments of the present invention embrace the following criteria be used for primer selection: [0051]a. For each primer at its 3' end, it matches any other primer in the set by less than 4 bp without gap, or 8 bp with a one-base gap; and in any portion of its sequence it complements any other primer by less than 9 bp without gap, or 12 bp with a gap. Alternatively stated, each primer of the primer pair complements less than nine contiguous bases or 12 contiguous bases with a gap of any other primer of the plurality of primers; [0052]b. The Tm of each primer is between 46° C. and 56° C.; [0053]c. The GC content of each primer is between 35% and 70%; and
[0054]d. Each primer at its 3' matches any but its own amplicon in the plurality of primers and probes by less than 14 bp without gap, or 18 bp with a gap. In other words, the 3' end of each primer complements at least 14 contiguous bases or 18 contiguous bases with a gap of the gene of interest.
[0055]When amplifying a plurality of amplicons, primers can be selected such that the lengths of all amplicons in the set differ by at least 7% so that the amplification products can be analyzed by gel electrophoresis when the gene set is small.
[0056]While a determination of whether a primer will interact with another primer or probe will minimize the interaction among the selected oligonucleotide sequences and amplicons, other DNA/RNA sequences present in the sample may also be a source of unexpected interaction. Therefore, the present method further includes the step of comparing the sequence of the pair of primers to sequences in one or more databases of genomic or mRNA sequences and determining whether the pair of primers will amplify a non-specific sequence in silico. This step is applied to check for interactions of the selected primers to any genomic and/or RNA sequences in the genome to ensure even higher amplification specificity during multiplex amplification. By way of illustration, primers are aligned to genomic and/or RNA sequences in databases using a program such as BLAST (Altschul, et al. (199) J. Mol. Biol. 215:403-410; Altschul, et al. (1997) Nucleic Acids Res. 25:3389-3402) and it is determined whether non-specific amplification could occur. Non-specific amplification is defined as alignment of the two primers with a non-target genomic or RNA sequence, in the correct orientation for amplification of an amplifiable product. Any primers resulting in non-specific amplification are removed from the set. After a primer is removed because of non-specific amplification, it can be maintained in a database as an "invalid" primer sequence, so that any subsequent primer selection steps will not select the primer again.
[0057]In addition to probes and pairs of primers, a nested primer is also selected for generating single-stranded DNA after multiplex amplification. As illustrated in FIG. 1A, the nested primer anneals to the amplicon created from the pair of primers and produces, in an amplification reaction, a single-stranded nucleic acid molecule hybridizable with the probe of the previous steps. The length, GC content, Tm and complementarity criteria of the nested primer are the same as those for the probe.
[0058]All possible frames of pair or primers and the nested primer flanking the probe are analyzed and the primer sequences with optimal Tm are selected. The method of the invention can also be used to design only pairs of primers for a list of genes of interest for multiplex amplification without microarray analysis.
[0059]Since the method of the invention selects primers and probes using stringent criteria, a user may list the genes to be included in the analysis in a priority order. In this respect, primers and probes can be designed based on the priority of the genes to ensure the inclusion of genes with higher priorities in the set. It is contemplated that primer and probe selection can start with a new set of genes or add new genes into an existing set. In most cases, multiple rounds of selection may need to be performed for a given set of genes of interest. Each round of selection may only select primers and probes for a fraction of the genes in the set. More round(s) of selection can be performed on top of the previous round to select primers and probes for more genes. The selection cycles can be repeated until a satisfactory number or all of genes are included, or no more genes can be added into the set through the selection.
[0060]In so far as the synthesis of oligonucleotide primers and probes is routinely practiced in the art, the primers and probes of the present invention can be synthesized by any conventional or routine method known to those skilled in the art. In addition, it is contemplated that the selection of probes and primers can be carried out by the skilled artisan or can be achieved using a computer executable program, wherein a list of genes of interest is provided as input and a plurality of probes and primers is provided as output.
[0061]Subsequent to primer and probe selection and synthesis, gene expression profile analysis is carried out by detecting and/or quantifying the amount of expression products in a sample, i.e., any substance containing nucleic acid material. As illustrated in FIG. 1A, the plurality of primers is used in an amplification reaction in the presence of a sample to produce double-stranded amplicons. The double-stranded amplicons are then contacted with the plurality of nested primers to amplify single-stranded nucleic acid molecules, which are then quantified via hybridization with the plurality of probes. As used herein, the terms "quantitating" or "quantifying" when used in reference to an amplification product, refers to determining the quantity or amount of a particular sequence that is representative of an expression product in the sample. For example, but without limitation, one may measure the intensity of the signal from a labeled probe. The intensity or quantity of the signal is typically related to the amount of amplification product. The amount of amplification product generated correlates with the amount of expression product present prior to amplification, and thus, in certain embodiments, is indicative of the level of expression for a particular gene.
[0062]Advantageously, the method of the present invention is highly specific. In contrast to the present invention, conventional high-throughput systems and methods fail to discriminate mRNA from other related DNA and RNA sequences. Using primers amplifying sequences across intron(s) and probes composed of sequences in adjacent exons is a critical enhancement to achieve such high specificity. Furthermore, all primer, probe and amplicon sequences are subjected to exhaustive searches against databases of the entire human genome and transcriptome to ensure these sequences are unique. Experimentally, when genomic DNA was used as samples, signals were only detected for only 2 or 3 genes (0.2%) out of the 1,135 genes. Based on previous studies, these signals may become undetectable in the presence of specific sequences which may compete out the nonspecific amplification.
[0063]In addition to being highly specific, the present method is also highly sensitive. It is known that multiplex amplification can detect >1,000 single-copy sequences simultaneously from single haploid sperm cells (Wang, et al. (2005) Genome Res. 15(2):276-283). The fact that >90% of these sequences are detectable indicates that with the specially designed primers herein, most, if not all sequences can be well-amplified in parallel with very limited, if any, interaction among the primers. Since the primers used for gene profiling are designed in the same way, it is reasonable to believe that most gene transcripts are also amplified in parallel. However, since the copy number of different gene transcripts in cells varies widely, the outcome of amplification would be different from that using single-copy sequences. When only single-copy sequences are used in multiplex amplification, most, if not all, sequences may reach the detectable amount before the system is saturated. However, when gene transcripts are amplified, whether a transcript reaches a detectable amount before the system is saturated depends on its copy number in the sample, and not all sequences may reach a detectable amount at the end of amplification. This is likely why some sequences are undetectable by microarray but detectable by gel assay.
[0064]Using the method of the invention, a total of 686 gene transcripts were detected from three single cells, which is comparable to 676 for the three 100-cell samples and 693 for all non-single-cell samples from the same cell line. The sensitivity of the method is further proved by the fact that results from 100-cell samples are very similar to each other and to those from 10,000 cells. In addition, specific gene expression profiles are obtained from different cell lines using as few as 100 cells.
[0065]The sensitivity of the method is further illustrated by the results showing that a significant portion of transcripts can not be detected from NCI/ADR-RES samples but are detected from the MCF-7 samples or single cell samples, and vice versa. This also indicates that low microarray intensities for these transcripts are not false negatives, and are either not present or present in very low abundance in the respective samples.
[0066]In contrast to conventional approaches, the present method is also very simple to carry out. Unlike other methods that involve multiple steps and use multiple enzymes, the method herein can be used to analyze a large number of expression products amplified by a single reverse transcription (RT)-PCR step directly from cell lysates without RNA extraction. In this manner, a large number of samples can be analyzed easily and cost-effectively. The simple experimental procedure is also the basis of the high degree of sensitivity since it avoids complicated RNA extraction and processing procedures before and during amplification, which may cause RNA degradation or loss.
[0067]Indeed, when working with RNA, extra precaution has to be taken to prevent RNA degradation. The instant method does not require RNA extraction; once cells are lysed, RNA is directly released to the RT-PCR buffer and used as template immediately. Thus, there is almost no chance for RNase to degrade the RNA templates.
[0068]Many studies require the analysis of only a subset of the genes in the human genome and often focus on different gene groups. Therefore, a flexible system is highly desirable. Using the instant method, a large number of expression products can be combined into a single multiplex group. Genes can be easily organized into different subgroups upon need, and can also be re-grouped at any time without altering the reaction conditions. New gene products can be added to an existed set easily.
[0069]The capacity of multiplex RT-PCR is another aspect of high-throughput gene expression profiling because it not only makes the amplification of a large number of gene products affordable and cost-effective, but it also eliminates challenges involved in quality control of RT-PCR for a large number of genes individually (Aguilar, et al. (2000) J. Clin. Microbiol. 38(3):1191-1195; Cerveira, et al. (2000) Br. J. Haematol. 109(3):638-640). However, the capacity of multiplex amplification is often limited by interaction between conventional primers. One study reported a screening of 29 expressed genes using multiplex RT-PCR, but was unable to reduce the number of the reaction tubes to less than eight (Pallisgaard, et al. (1998) Blood 92(2):574-588). Other studies achieved multiplexing of up to nine genes with nonspecific RT primers (Malhotra, et al. (1998) Nucleic Acids Res. 26(3):854-856; Tietjen, et al. (2003) Neuron 38(2):161-175). Studies using multiple sets of gene-specific primers in single reactions have also been reported, but none of these generated enough products for the analysis of all expressed genes in the samples (Cerveira, et al. (2000) supra; Clipsham & McCabe (2001) Mol. Genet. Metab. 74(4):435-448). In the present study, success with multiplex RT-PCR for 1,135 mRNA species was achieved. Such a success was based on a combination of several technological developments, including computerized primer design with predicted minimal interaction, a narrow primer Tm range, small amplicon sizes, and optimization of amplification conditions. With the current method, it is possible to include two thousand or more gene transcripts in a single multiplex amplification group, and to analyze all human gene transcripts using several multiplexing amplification groups. After pooling amplified products from the multiplexing groups, all genes can be analyzed with a single microarray. With this system, large-scale gene expression profiling becomes highly affordable and cost-effective. If the primers and probes used in the high-throughput analysis are made accessible to the research community through a distribution system, large- and genome-scale gene expression profiling can become even more affordable and cost-effective.
[0070]Drug resistance to a broad spectrum of chemotherapeutic agents is a major obstacle in the clinical treatment of human cancer. Therefore, understanding the underlying mechanisms, together with detection of altered gene expression patterns with very sensitive and reliable techniques, is of great importance in increasing the efficacy of cancer therapy. An embodiment of the highly sensitive gene expression profiling system in cancer research is analysis of disseminated tumor cells. Analysis of individual cells is necessary for understanding the early dissemination of tumor cells from a small number of cells or single primary tumor cell. Disseminated tumor cells remaining untouched after complete resection of the primary tumor currently can be detected by bone marrow aspirates. With the highly sensitive method of this invention genetic signature in these cells can be detected, which will provide molecular basis for new therapeutic targets. For example, erbB2 expression has been found to be a therapeutic target for metastatic breast carcinoma. Direct identification of mRNA like that of erbB2 in micrometastatic cells can facilitate the development of an effective therapy, preventing the development of incurable metastasis. In addition, successful analysis of mRNA from microdissected frozen tissue sections without RNA isolation has been demonstrated with human tissue. As such, this invention will be very useful for global gene expression analysis of a biopsy specimen. In addition to cancer research, multiplex RT-PCR with single cells finds application in the study of molecular neurophysiology. This invention can be used to examine the expression of many genes within individual neurons or other cells that can also be characterized with regard to their morphological, electrophysiological and pharmacological features.
[0071]The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example hereinbelow. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as A Laboratory Manual, and Molecular Cloning (from Cold Spring Harbor Laboratory Press); Stryer, Biochemistry, (W H Freeman); and Gait, Oligonucleotide Synthesis: A Practical Approach, 1984 (IRL Press, London).
[0072]The present invention also contemplates many uses for oligonucleotides attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping, and diagnostics. Gene expression monitoring, and profiling are described, e.g., in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore are described in U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460 and 6,333,179.
[0073]The invention is described in greater detail by the following non-limiting examples.
Materials and Methods
[0074]Cell Lines and Single Cell Preparation. Human breast cancer cell line MCF-7 and ovarian cancer cell line NCI/ADR-RES are known in the art (Wu, et al. (2003) Cancer Res. 63(7):1515-1519). The cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37° C. in a humidified atmosphere containing 5% CO2. After counting with a hemacytometer, cells were suspended in PBS (phosphate-buffered saline) to 1000 cells/μl or other desirable densities. Two μl was dispensed into an EPPENDORF tube containing cell lysis buffer (1.5 μl RNase inhibitor, 4 μl of 5× QIAGEN ONESTEP RT-PCR buffer, 12.5 μl H2O). Single cells were prepared from a diluted cell suspension of 2 cells/μl in 1×PBS. About 0.5 μl of the suspension was pipetted onto a small piece of glass coverslip, and was checked under a microscope. If the droplet contained only one cell, the piece of the coverslip was then transferred into an EPPENDORF tube containing the cell lysis buffer. The tube was immediately frozen in an ethanol/dry ice bath and stored at -80° C. until use.
[0075]Selection of Genes for mRNA Profiling. Genes used in the present study were selected for their involvement in fundamental cell functions such as cell cycle, apoptosis, cell matrix, DNA repair, DNA replication, somatic recombination, RNA transcription and regulation, and protein translation and regulation. A complete list of the 1,135 genes of interest and their accession numbers is described by Hu et al. (2008) BMC Genomics 9:9. The borders between exons and introns for the selected genes were determined by aligning of the mRNA to genomic sequences using the BLAT program maintained by the University of California, Santa Cruz.
[0076]Primer and Probe Design. A computer program was developed to select primers and probes according the criteria described herein. Each oligonucleotide probe for microarray analysis was designed to be composed of sequences of two adjacent exons to specifically interrogate the cDNA from corresponding mRNA sequence, but not the corresponding genomic sequences or cDNA from unprocessed RNA. To facilitate microarray analysis, the 3'-ends of all probes terminated before a "G" in the template sequence so that they could be labeled with the same fluorescent color by incorporating fluorescently labeled Cy5-ddCTP. The lengths of the probes ranged from 2.2 to 31 bases, and the GC-content of the probes ranged from 30% to 70% with their Tm's from 54.4° C. to 65.2° C. A representative list of probes is provided in Table 1.
TABLE-US-00001 TABLE 1 Accession Tm SEQ Gene Symbol No. Probe (° C.) ID NO: EBF4 AB037863 CGGCCGCTTTGTCTACACAGCT 59.9 1 NCOA3 AF012108 AGTTTCTCATGGCACTCAAAATAGGCCT 59.6 2 BAG1 AF022224 GCCGGGTCATGTTAATTGGGAAAAAGAA 59.1 3 MAP2K7 AF022805 TCCAGTCCTTCGTCAAAGACTGCCTTA 60.2 4 PRY2 AF039843 CAACGGGTCGCAGCCCTTGCTG 63.1 5 IGFBP5 AF055033 TGAGACAGGAGTCTGAGCAGGGC 60.3 6 SCGB2A1 AF071219 CTCCTCCTGCACTGCTATGCAGATT 59.3 7 SERF1A AF073519 GGCAGTCAAGCTATCCTCTTTCCTCTTT 59.3 8 MS4A7 AF201951 CTCTGGAAAACAATCAACTAAGCCCTTTGAC 59.2 9 PECI AF257175 ACTCTACGCGCTATATAAGCAGGCCA 60.1 10
[0077]Each pair of PCR primers was designed to amplify sequences in two adjacent exons flanking a large intron and to ensure specific amplification of the desirable mRNA sequences rather than the respective gene or unprocessed RNA sequences. To enhance the amplification specificity, the program always searched for candidate amplicon sequences separated by large introns in each gene. The melting temperatures (Tm's) for all selected primers ranged from 50.1° C. to 61.6° C., and the GC-contents ranged from 32% to 70%. The lengths of the amplicons ranged from 72 to 150 bases. An illustrative list of primer pairs is provided in Table 2.
TABLE-US-00002 TABLE 2 Amplicon Tm Length SEQ Gene Symbol Primer (° C.) (bp) ID NO: EBF4 L, AGCAGTTTTGCAAGGGATGCC 56.5 84 11 R, GTAGCCTCTGGAATCCGTAGTCAA 56.2 12 NCOA3 L, GCATGTTGTCCATGGAACAAGTTTC 55.8 118 13 R, GCTCTTTCGTCACTCTGGCCT 56.8 14 BAG1 L, TACAAGATGGTTGCCGGGTCA 56.3 93 15 R, CAGACTTCTCCAAATGTTTCAACTT 52.6 16 MAP2K7 L, CACATGGGCTTCTCGGGGGA 58.7 120 17 R, CGTCTCGTAGCGCTTGATGAAG 55.6 18 SPRY2 L, GCCAGAGCTCAGAGTGGCAA 56.9 102 19 R, CTGGGTGAGGGCGTCTCTGG 58.8 20 IGFBP5 L, CGGATCATCTCTGCACCTGAGA 56.0 88 21 R, CTTTGAGCTCCTGCAGGGAAG 55.3 22 SCGB2A1 L, CTCATGCTGGCGGCCCTCCT 61.6 80 23 R, ATGGTCTTTTCAACCATGTCCTCC 55.6 24 SERF1A L, CTGCTTTCTCTGAGAGGCAGTCA 56.6 87 25 R, GCCCGCCAGAAAAACATGAAGA 56.4 26 MS4A7 L, GGATCCCTCTCAATTATCTCTGGAA 54.1 92 27 R, CCTGCAGTAACAGAACTCACTGC 55.9 28 PECI L, CAGGAAACGAAGTGAAGCTAAAACT 54.3 90 29 R, AGTCAAATACACCTGGTTTGGGC 55.9 30 L, left primer; R, right primer.
[0078]Using the same melting temperature and GC content criteria as for the probes, nested primers were selected. Nested primers were selected for annealing to the amplicons generated by the pairs of primers and for also generating a molecule that would hybridize to probes. An illustrative list of nested pairs is provided in Table 3.
TABLE-US-00003 TABLE 3 Tm SEQ Gene Symbol Primer (° C.) ID NO: EBF4 TCCGTAGTCAATGGTGGGCTCGTT 55.6 31 NCOA3 GGCCCAACAAGATCATCCAGGGAA 55.7 32 TTC BAG1 CAACTTCTTTAGTTCAACCTCTTC 55.0 33 CTGTGGA MAP2K7 AGCTTATTATACTTTGGTCTCTTC 52.5 34 CTGTGAT SPRY2 CCCACGCTGTCTGCCACCGTCA 58.6 35 IGFBP5 GGAAGCCTCCATGTGTCTGCGG 54.9 36 SCGB2A1 TCTTTTCAACCATGTCCTCCAGGA 55.2 37 GTTTG SERF1A AGAAAAACATGAAGAAAACCCAGG 51.6 38 AAATTAG MS4A7 TCACTGCATTTGAGGTCAAGCTGC 54.9 39 T PECI TGGTTTGGGCATGTTACAAGGTCC 54.9 40 TT
[0079]All primers and probes were subjected to interaction analysis with a computer program developed for designing high-throughput multiplex nucleotide acid detection and all primers were subjected to analysis to determine whether they would amplify an amplifiable product in silico. Probes complementary to intron regions of some genes were also designed as negative controls. All amplicon sequences were subjected to BLAST search to ensure their uniqueness.
[0080]Gene-Specific Reverse Transcription and Multiplex RT-PCR. Cells in the lysis buffer described above were lysed with three repeating cycles of alternating one-minute incubations from the ethanol/dry ice mix to a 37° C. water bath before RT-PCR. One-step RT-PCR was carried out in a 50-μl reaction containing primers (20 nM each) for all the 1,135 mRNA species, 2.5 mM MgCl2, the four dNTPs (400 μM each), and 2.0 μl QIAGEN ONESTEP RT-PCR Enzyme Mix without degenerated primers. The samples were first incubated at 50C for 40 minutes for cDNA synthesis, and then were heated to 95° C. for 15 minutes to inactivate the reverse transcriptase and activate the Taq DNA polymerase followed by 45 PCR cycles. Each PCR cycle included 40 seconds at 94° C. for denaturation, and 1 minute at 55° C. and 5 minutes of ramping from 55° C. to 70° C. for annealing and extension. A final extension step was carried out at 72° C. for 3 minutes at the end of the PCR. All PCRs were performed with the PTC100 Programmable Thermal Controllers (MJ Research). Single-stranded DNA (ssDNA) was generated using the same conditions as in multiplex PCR. Only one primer for each sequence was used, and 40 thermal cycles were carried out.
[0081]RT-PCR with Individual Gene Transcripts. RT-PCRs with individual gene transcripts were performed for a group of genes with different amounts of signal intensities detected from the two cell lines, NCI/ADR-RES and MCF-7. For each gene, an aliquot (equivalent of 100 cells) from the same cell lysate used for multiplex gene expression profiling was used. Conditions for one-step RT-PCR were similar to those for multiplex one-step RT-PCR. mRNAs transcribed from β-actin and α-tubulin genes served as internal controls. The PCR products were assayed by gel electrophoresis. Gels were imaged using an Image Station (Model 440, KODAK, New Haven, Conn.). Gel band intensities were digitized with the software, KODAK 1D 3.5.
[0082]Microarray Design, Hybridization, and Probe Labeling by Single-Base Extension Assay. Oligonucleotide probes were printed onto glass slides in duplicate with a spot diameter of 160 μm and a center-to-center distance of 250 μm by using the OMNIGRID Accent Microarrayer (Gene Machines, Calif.). One hundred fourteen spots with only microarray printing buffer without probes were used as negative controls and were distributed spatially evenly across each array. Microarray analysis was performed according to established methods (Wang, et al. (2005) supra).
[0083]Microarray Scanning and Data Analysis. Microarrays were scanned with a GENEPIX 4000 scanner (Axon Instruments, Foster City, Calif.). The resultant images were digitized with the accompanying software GENEPIX Pro (version 4.0). The mean values of the signals from the duplicate spots of each probe were used for the analysis herein. Background signal was determined by using negative control probes that were complementary to the intron sequences of the corresponding genes or random sequences, and was subtracted from the sample signals. For the comparative expression analysis of the cell lines MCF-7 and NCI/ADR-RES, the array data were normalized by the Lowess smoothing method (Yang, et al. (2002) Nucleic Acids Res. 30(4):e15; Quackenbush (2002) Nat. Genet. 32 Suppl:496-501). After background subtraction, genes with negative intensity values in both duplicated samples were excluded for further analysis. The log ratios of the intensities of the remaining genes in two cells lines were used to make calls and to identify the differentially expressed genes in the samples.
Cancer Gene Expression Array
[0084]To establish a cancer gene expression array, a panel of cancer-related genes were selected based on their known functions and/or cancer-associated expression patterns from published literature. All amplicon sequences were subjected to computational screening to ensure their uniqueness. Primers and probes were selected according to a series of criteria described herein. Most primer pairs amplify sequences in two neighboring exons separated by large introns. The intron lengths ranged from 79 bp to 90 kb with an average of 2.0 kb and 97% of the introns are longer than 200 bp. Initially 1,445 genes were used as the input for the primer and probe design program. Primers and probes for 1,120 (77.5%) of these genes were selected. The remaining 22.5% failed in the selection process because of lack of introns or suitable sequences for primers and/or probes. Fifteen of these remaining genes with important functions in cancer development were included in the panel. Primers and probes were designed based on the unique sequences in these genes, and were not required to have introns internally located within the amplified sequences. Therefore, a total of 1,135 genes were included in the multiplex assay.
[0085]Microarray-based single-base extension (SBE) assay has been used to genotype single nucleotide polymorphisms (SNPs) (Wang, et al. (2005) supra; Greenawalt, et al. (2006) Genome Res. 16(2):208-214; Hu, et al. (2006) Nucleic Acids Res. 34(17):e116). In the present study, SBE was adapted for gene expression profiling. To simplify the analysis, all probes were designed to terminate immediately before a `G` site in the templates. In this way, the probes were extended by a single base, dideoxynucleoside triphosphate (ddCTP) that was fluorescently labeled. By using one color, the bias associated with different dyes was also eliminated.
[0086]To test the reproducibility of the system, gene expression profile analysis was conducted for three duplicated 100-cell samples from an ovarian cancer cell line, NCI/ADR-RES (Liscovitch & Ravid (2007) Cancer Lett. 245(1-2):350-352) and two 100-cell samples from a breast cancer cell line, MCF-7. The data indicated that 660 (58.2%), 663 (58.4%), and 662 (58.3%) gene transcripts were detected from the three 100-cell samples of NCI/ADR-RES, respectively. Of these transcripts, 650 (>98%) were detected from all three duplicates. Signal intensities for the 1,135 genes were strongly correlated between the duplicates (Pearson's r=0.977, 0.974, and 0.949, respectively). Of the 650 transcripts detected in all three NCI/ADR-RES 100-cell samples, only 6 (0.9%), 17 (2.6%), and 1 (0.2%) transcripts had their signal intensities differing by >2 fold between each two of these three duplicates. Twenty-six transcripts were detected from only one or two of the three samples. The signal intensities for these transcripts were low. Only one transcript in one sample had its signal intensity >1,000, indicating that the inconsistence among the duplicates was due to low signals of these transcripts.
[0087]For the two 100-cell samples from MCF-7, 615 (54.2%) and 614 (54.1%) gene transcripts were detected, respectively, with 597 (>97%) detected in both. Of these 597 transcripts, 562 (94.1%) had signal intensities differing less than two fold. Similar to the situation with NCI/ADR-RES samples, all 34 transcripts that were detected in only one sample but not the other had low signal intensities with only nine genes whose signal intensities were >1,000 in one of the two samples.
[0088]Because samples prepared from a large number of cells are usually associated with high reliability, the microarray results of the NCI/ADR-RES 100-cell samples were further compared with those from a 10,000-cell sample of the same cell line. Resulting data also showed a high degree of correlation (r=0.961). This analysis indicated that 630 (96.7%) of the 650 gene transcripts detected from all the 100-cell samples were also detected from the 10,000-cell sample. Sixty-three gene transcripts were detected in at least one of the three 100-cell samples but not in the 10,000-cell sample, or vice versa. Of these 63 gene transcripts, 61 had signal intensities below 1,000 in all the three 100-cells. However, the change from 100 to 10,000 cells did enhance the detection of 21 gene transcripts whose signal intensities were >2 fold greater in the 10,000-cell sample than those in the 100-cell samples. Among these 21 transcripts, six had signal intensities in the 10,000-cell sample more than 15 fold greater than the average intensities of the corresponding genes in the three 100-cell samples, indicating that using 10,000 cells may have significantly increased the copy numbers of these transcripts or changed their absence status to presence. These data indicated that the system not only could produce very reliable results even with as few as 100 cells but also was very sensitive to the copy number change for the low-copy-number gene transcripts.
[0089]To further test the sensitivity of the high-throughput gene expression profiling system, single NCI/ADR-RES cell samples were prepared and used for multiplex gene expression assay of the 1,135 mRNA species. The numbers of gene transcripts detected from the three single-cell samples were 590, 576, and 614, respectively. Of these transcripts, 507 were detected from all single cells. Of the 503, 463 (92.0%) were also detected from all non-single-cell (100-cell and 10,000-cell) samples, indicating a prevalent expression of these genes in most, if not all, cells at relatively high levels.
[0090]A wider range of gene transcripts was detected from the three single-cell samples compared to the non-single-cell samples. Four hundred forty-nine transcripts were undetectable in all three single cell samples, a number which is not greater than that (459) for the three 100-cell samples and is comparable to that (442) for all non-single-cell samples. The number of undetectable gene transcripts from all single and non-single cell samples is 357. This number means that from single cells, a comparable number of genes were not only detected, but also a new set of 92 genes were detected that could not be detected with non-single-cell samples of the same cell line.
[0091]The robustness of gene expression profiling with single-cell samples was also demonstrated by the signal intensities. As described above, most transcripts that were detected from some but not all non-single-cell samples had low signal intensities with very few >1,000. The scenario with single cells was very different. Of the 503 gene transcripts detected from all single cells, 40 were detected in one to three non-single-cell samples but not all four. All 40 but one had signal intensity >1,000 in at least one of the three single-cell samples. Of the 183 transcripts that were only detected from one or two single-cell samples, 108 (59.0%) had signal intensity >1,000. The strong and robust signal intensities detected from single-cell samples indicated that the method was very sensitive.
[0092]Unlike the gene transcripts detected from all non-single-cell samples which account for more than 95% of gene transcripts detected from each of these samples, the 503 gene transcripts detected from single cells only account for 85. 3%, 87.3% and 81.9% of the transcripts detected from individual single-cell samples, respectively. Pairwise comparison of the results from the single-cell samples yielded correlation coefficients of 0.780, 0.700, and 0.711, respectively compared with 0.949 or greater for other non-single-cell samples. From all single and non-single-cell samples, 778 gene transcripts were detected, of which 315 (40.5%) were detected from some but not all samples. This is in contrast with the scenario of non-single-cell samples from which gene transcripts that were only detected from some but not all samples were a very small portion. Furthermore, of these 315 transcripts, 177 (56.2%) were either detected from only single cells or from non-single cell samples.
[0093]The high degree of concordance among the results from the non-single-cell samples, and the significant differences among those from single cells, and between single cells and non-single-cell samples indicate that most, if not all, of these differences are real. As mentioned above, this is further supported by the robustness of the signal intensities detected from single-cell samples for the gene transcripts that were detected differently between the single cells and non-single-cell samples. It is conceivable that heterogeneity in clonality and/or genetic alterations in the cells of a cell line could be major factors contributing to the differences. In addition, a considerable portion of the cells may be at different cycle stages during which groups of genes are expressed differently. Therefore, while gene expression in single cells could differ in various aspects, 100 cells may well represent the entire cell population because, after all, the cell line cells are from the same tissue and the same donor. Therefore, genes that are detectable in a cell population may not be expressed or expressed at very low levels in certain single cells. Conversely, genes that are detectable in particular single cell samples may not be expressed or expressed at very low levels in the majority of the cell population.
[0094]When the gene expression profiles of NCI/ADR-RES were compared with those of MCF-7, a considerable number of genes were shown to be expressed differentially in these two cell lines. Of the 1,135 gene products, 531 (46.8%) were detected from samples of both cell lines (not including single cell samples). Seventy-five gene transcripts were detected in all NCI/ADR-RES non-single-cell samples, but not in the MCF-7 samples, and 43 were detected in the opposite way.
[0095]The specificity of the high-throughput gene expression method was demonstrated by the results from different cell line samples and by those from different single cells as described above. To further demonstrate the specificity of the system, human genomic DNA samples were amplified with the same multiplex RT-PCR procedure and analyzed by microarray. Very few probes (<0.2%) were shown to have signals above the background, indicating that the method was very specific and could discriminate between the target mRNA sequences from their genomic counterparts, and therefore, the unprocessed transcripts.
Sequence CWU
40122DNAArtificial SequenceSynthetic oligonucleotide 1cggccgcttt gtctacacag ct 22228DNAArtificial SequenceSynthetic oligonucleotide 2agtttctcat ggcactcaaa ataggcct 28328DNAArtificial SequenceSynthetic oligonucleotide 3gccgggtcat gttaattggg aaaaagaa 28427DNAArtificial SequenceSynthetic oligonucleotide 4tccagtcctt cgtcaaagac tgcctta 27522DNAArtificial SequenceSynthetic oligonucleotide 5caacgggtcg cagcccttgc tg 22623DNAArtificial SequenceSynthetic oligonucleotide 6tgagacagga gtctgagcag ggc 23725DNAArtificial SequenceSynthetic oligonucleotide 7ctcctcctgc actgctatgc agatt 25828DNAArtificial SequenceSynthetic oligonucleotide 8ggcagtcaag ctatcctctt tcctcttt 28931DNAArtificial SequenceSynthetic oligonucleotide 9ctctggaaaa caatcaacta agccctttga c 311026DNAArtificial SequenceSynthetic oligonucleotide 10actctacgcg ctatataagc aggcca 261121DNAArtificial SequenceSynthetic oligonucleotide 11agcagttttg caagggatgc c 211224DNAArtificial SequenceSynthetic oligonucleotide 12gtagcctctg gaatccgtag tcaa 241325DNAArtificial SequenceSynthetic oligonucleotide 13gcatgttgtc catggaacaa gtttc 251421DNAArtificial SequenceSynthetic oligonucleotide 14gctctttcgt cactctggcc t 211521DNAArtificial SequenceSynthetic oligonucleotide 15tacaagatgg ttgccgggtc a 211625DNAArtificial SequenceSynthetic oligonucleotide 16cagacttctc caaatgtttc aactt 251720DNAArtificial SequenceSynthetic oligonucleotide 17cacatgggct tctcggggga 201822DNAArtificial SequenceSynthetic oligonucleotide 18cgtctcgtag cgcttgatga ag 221920DNAArtificial SequenceSynthetic oligonucleotide 19gccagagctc agagtggcaa 202020DNAArtificial SequenceSynthetic oligonucleotide 20ctgggtgagg gcgtctctgg 202122DNAArtificial SequenceSynthetic oligonucleotide 21cggatcatct ctgcacctga ga 222221DNAArtificial SequenceSynthetic oligonucleotide 22ctttgagctc ctgcagggaa g 212320DNAArtificial SequenceSynthetic oligonucleotide 23ctcatgctgg cggccctcct 202424DNAArtificial SequenceSynthetic oligonucleotide 24atggtctttt caaccatgtc ctcc 242523DNAArtificial SequenceSynthetic oligonucleotide 25ctgctttctc tgagaggcag tca 232622DNAArtificial SequenceSynthetic oligonucleotide 26gcccgccaga aaaacatgaa ga 222725DNAArtificial SequenceSynthetic oligonucleotide 27ggatccctct caattatctc tggaa 252823DNAArtificial SequenceSynthetic oligonucleotide 28cctgcagtaa cagaactcac tgc 232925DNAArtificial SequenceSynthetic oligonucleotide 29caggaaacga agtgaagcta aaact 253023DNAArtificial SequenceSynthetic oligonucleotide 30agtcaaatac acctggtttg ggc 233124DNAArtificial SequenceSynthetic oligonucleotide 31tccgtagtca atggtgggct cgtt 243227DNAArtificial SequenceSynthetic oligonucleotide 32ggcccaacaa gatcatccag ggaattc 273331DNAArtificial SequenceSynthetic oligonucleotide 33caacttcttt agttcaacct cttcctgtgg a 313431DNAArtificial SequenceSynthetic oligonucleotide 34agcttattat actttggtct cttcctgtga t 313522DNAArtificial SequenceSynthetic oligonucleotide 35cccacgctgt ctgccaccgt ca 223622DNAArtificial SequenceSynthetic oligonucleotide 36ggaagcctcc atgtgtctgc gg 223729DNAArtificial SequenceSynthetic oligonucleotide 37tcttttcaac catgtcctcc aggagtttg 293831DNAArtificial SequenceSynthetic oligonucleotide 38agaaaaacat gaagaaaacc caggaaatta g 313925DNAArtificial SequenceSynthetic oligonucleotide 39tcactgcatt tgaggtcaag ctgct 254026DNAArtificial SequenceSynthetic oligonucleotide 40tggtttgggc atgttacaag gtcctt 26
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Patent applications by Guohong Hu, Piscataway, NJ US
Patent applications by Honghua Li, Monmouth Junction, NJ US
Patent applications in class Involving nucleic acid
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wikiHow to Draw an Abalone
Abalone are so colorful, especially in reds greens and blues. Carefully shape the shell to include the proper line of holes that are distinct to their filtering system.
1Prepare your drawing by keeping in mind the areas of color will overlap. First create a simple scenery showing sand and water. Using a soft brown pencil start drawing the rounded lip for the front of your abalone.
2Bring up your lines along each side and diminish the size gradually to the back of the shell. Lightly swirl a line to draw together the small end of the shell. Each growth section to the back of the shell are drawn next with soft broken lines. Decrease the size of each section back to the beginning of the swirl. The shell is not very large in size in real measurement maybe 3 to 4 inches (7.6 to 10.2 cm). Along the left side of the shell create holes that start in the front and gradually get smaller as they follow in a line to the smaller end of the shell.
3Add more colors with more vibrancy. Carefully blend areas of color with pastel chalk. Pick chalk one shade brighter than your pencil and fill in particular areas that show the curve of the shell, bringing out the areas around the holes and the swirl as well as the edges.
4Fill in the lightly colored areas with a heavier pencil of the same colors you began with to darken some of the concave shapes for depth. It should be looking pretty good and lively.
5Put some more color around the background closest to the shell with a little amber and gold for contrast. Now, all that is needed a spray fixative for the pastel and your signature on the page.
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- A textured paper is the better choice for this simple design.
- Do not overspray your fixative, A light coating is sufficient.
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A pre-recorded streaming VIDEO replay of the October 2015 webcast, The Employment Law Docket of the 2015-2016 U.S. Supreme Court Term: An Analysis of the Employment Issues That the Supreme Court Will Decide Next Year .
The Supreme Court's decisions in next year's term promise to have significant ramifications for employees, employers, and benefit plans. During this interactive program, the presenters will provide an advance view of the employment issues that the Supreme Court will decide in its new term.
The speakers will discuss issues of class action procedure, mandatory employment claim arbitration, EEOC litigation prerequisites, privacy, ERISA fiduciary claims, and other issues that will be featured on the Supreme Court's 2015-2016 docket.
Save up to 40% on online seminars and webcasts with the purchase an 8- or 12-credit Online Bundle. Take up to 365 days to choose the programs you want.
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James P. McElligott Jr., McGuireWoods / Richmond James McElligott advises and defends private and publicly held corporations, public agencies, benefit plans, and fiduciaries concerning employee benefits, executive compensation, and related labor and employment issues, including ERISA litigation, class actions, claims for employee benefits and executive compensation, breach of fiduciary duty (including stock-drop class actions), multiemployer plans and withdrawal liability, retirement medical, benefits in bankruptcy, and Pension Benefit Guaranty Corporation issues. Mr. McElligott is a fellow of the College of Labor and Employment Attorneys. He regularly publishes and lectures on employment, benefits, and compensation issues. He has been interviewed by the Wall Street Journal, New York Times, Baltimore Sun, Daily Labor Reporter, Pension and Benefits Week, Computer Law, Tax Report, CBS Business Watch, Business Insurance, Chicago Sun Times, Richmond Times-Dispatch, MicroSoft Network, WashTech.com, Employment and Labor Lawcast, and other publications.


text:
AUD/CAD: sell targets - 0.9400 and 0.9350
By: Dmitriy Chernovolov
- AUD/CAD reached sell target 0.9450
- Next sell targets - 0.9400 and 0.9350
AUD/CAD has been under strong bearish pressure lately – falling inside the minor impulse wave (iii) – which belongs to the 3rd impulse wave 3 - which started earlier , when the pair reversed down from the strong resistance level 0.9750 (as you can see from the daily AUD/CAD chart below). The price earlier reached the support level 0.9450, which was set as the sell target in our previous forecast for this pair.
AUD/CAD is likely to fall further inside the accelerated impulse waves (iii) and 3 toward the next sell targets at the support levels 0.9400 (low of waves 1 and (b)) and 0.9350.


text:
But the Dresdner Christstollen, or Dresden Christmas cake, existed long before those eighteenth-century flamboyancies. The Dresden Christmas market, or Striezlmarkt, held annually since 1534, is named after an old word for the Stollen, first mentioned inthe early fourteenth century.
The Dresden cake contains rum-soaked raisins, candied fruit, and almonds. It even became a symbol of East German pride, one of the few objects easterners were happy to send to western relatives.
After the collapse of the East German regime in 1989, sales plummeted. People were reluctant to spend money on the same old east German products now that they could get glossy western items that had been inaccessible before. For west Germans, too, there
was no longer the same buzz from the plain package from the severed east.
In the past two years, however, there has been a turnaround, and rediscovered pride. Manfred Gopfert, spokesman for the Dresden bakers, says the market for Dresden Stollen is now "bigger than ever - not just in Germany, but worldwide".
A Stollen Protection Association has been created. Every week "Stollen experts" meet and cakes are cut apart, scrutinised, sniffed, and tasted, before receiving the seal of approval.
Even more importantly, the Dresden bakers have launched a battle in the courts to ensure that only cakes from Dresden may in be called Dresden cakes - not the imitations now produced in Munich or Cologne. One Dresdener argued: "The cakes from the west all look so nice - but there's nothing real in them. They're worth nothing. You can't put a BMW badge on your car if you're driving a Volkswagen, can you?"
- More about:
- Cake (dessert)
- East Germany
- Germany
- Saxony


text:
Anyone have a 20 year old with CF?
I have a 23 yr old son with CF - diagnosed at 17 1/2. Has taken 5 years to get him to realize what living with this disease is all about. Still will not eat enough food and...
Started by Gayla on 09/09/2009 in Cystic Fibrosis Moms
Last update on 11/06/2012 by GIna


text:
The best letter you've ever read: Former nurse destroys archbishop's anti-gay viewpoint
23 comments:
Anonymous says
From a Cradle Catholic:
BRAVO! United we stand
Natalie Kirksey says
Wow! This was absolutely refreshing to read. As someone who identifies with the LGBTQ community, I most appreciate the people of this world who are not afraid of standing up for their fellow man and addressing the real, burning issues of the times.
Natalie Kirksey says
Wow! This was absolutely refreshing to read. As someone who identifies with the LGBTQ community, I most appreciate the people of this world who are not afraid of standing up for their fellow man and addressing the real, burning issues of the times.
Anonymous says
I hope this letter was sent to each of the Cardinals - it deserves translations and extra readings by all sorts of church-going "good people" - and there are some of those around.
Anonymous says
This letter is amazing...I amglad that someone is finally speaking out.
Dylan. Steele says
Thank you! it is about time someone addressed church descrimination on same sex marriages. thank you this made my day:)
Anonymous says
I am one of those that sat on the church and listen to the hate and unkind words. I thank you with all my hard for your living letter! Thank U so much! There is hope and love!
Anonymous says
I think most, if not all of the priest are using the Church to hide their own homosexuality. Maybe they should clean out their closets!!!
Adam farmer says
Excellent. But something tells me that sweet woman got no response....
Kayla Mitchell says
Jesus came to this earth to follow the old testament. to show other that if He could do it, ANYONE could do it. With that said it is STILL an ABOMINATION To God and MY Lord and Savior Jesus Christ. God does not accept homosexuality or NEVER WILL. The Holy Bible says God is the same YESTERDAY, TODAY AND FOREVER. Just because we live in this century does not make it an exception. Jesus loves you but he hates your SIN. He wants you to repent or SADLY, YOU WILL, go to hell whether you want to believe it or not. Now is the time to repent. if you cant leave homosexuality ask God. Tell Him your frustrations. The catholic church CANNOT and WILL NOT save you. PURGATORY is NOT REAL. the Bible states, in fact JESUS states that you shall not call a priest Father bc you have only ONE father who is in heaven. You should not believe or trust the priests, after all, the are the ones who killed Him, who crucified Him. Jesus said, I am the WAY, the TRUTH, and the LIFE, NOONE can come to the Father EXCEPT THRU ME. not the catholic church, not purgatory. So again I tell you repent before its too late. Jesus Christ is coming very soon and many, even HOMOSEXUALS, will stay here on earth and suffer the consequences of eternity in the Lake of FIRE and torment, the gnashing of teeth and where the worm NEVER dies!!!!
Kayla Mitchell says
oh and one more thing.don't think i'm bashing ya'll. i love ya'll with the love of Christ. This was just a message letting you know there is a way out, Jesus is the way out.
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Anonymous says
The nurse gets my vote aswell. Reading that's put my faith back in people, everyday people. I agree, don't use god to incite hate, love and get some insight. Knowing people like you are around inspires hope, all the best.
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Whether you're looking for a smaller venue for an executive retreat or facilities for a large conference, our meeting offers provide you with options to get the most value from your next event. To learn more about the possibilities, please contact our Sales Coordinator at 207.361.6207 or e-mail firstname.lastname@example.com.
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text:
|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|Steve Holdener <ste...@mail.amnesiac.net>
|Re: W2000 SP1 Delete of directory fails
|Wed, 06 Feb 2002 16:47:55 GMT
Martin van Wieringen wrote: > When I have a build.xml file which creates the build directory, compiles > the classes to the build directory then put the .class file on a jar file > in the dist directory and at the end deletes the entire build directory. It > works fine however if I envoke the build two times after each other then it > W2000 seems to lock the build directory so that the second build result in > failure when it tries to recreate the directory. I saw similar behavior on my W2000/SP2 box yesterday. I closed all open apps, and it still wouldn't delete the directory ("Access Denied"), so I rebooted. I suppose this is a workaround for now...that, or don't use Windows. < drifts off in a daydream about Linux, smiles wistfully > BTW, after Ant attempts to delete the directory, the dir appears stuck in some strange limbo state. It will show up in the explorer, but I can't even select it without getting the error. IIRC, I was not able to see it from a command prompt. I've not been able to reproduce this today, but I'll let you know if I can get any more details. -Steve -- To unsubscribe, e-mail: <mailto:email@example.com> For additional commands, e-mail: <mailto:email@example.com>
|View raw message


text:
Percy Champagne, former vice-chair of the board of directors for Riverside Health Care Facilities Inc., will be chair for the next year.
Craig Sanders stepped down from the position, as per the bylaw limiting a term as chair to three years.
"It's been a good three years," Sanders said yesterday.


text:
Bruce Hoppe, PhD, creator of Webwhompers, teaches Web Science in the College of Arts and Science at Boston University.
Dr. Hoppe is founder and president of Connective Associates LLC, where he has helped businesses large and small to improve productivity, innovation, and leadership with a network perspective. He writes a popular business blog Connectedness about social network analysis. Dr. Hoppe is a member of Harvard Law School's Social Physics project on Internet & Society, and previously was a member of MIT Sloan's Innovative Collaborative Knowledge Networks project.
As an experienced community leader, Dr. Hoppe often helps catalyze collective awareness and action. He works closely with the Leadership Learning Community, a nationwide collaborative of leadership development practitioners, funders, and thought leaders dedicated to social change.


text:
CBC Poetry Prize
The elegance and power of poetry
There are five names on the shortlist for this year's CBC Poetry Prize. Before we announce the winner, we want to introduce you to the finalists and their poetry.
Pamela Porter talks about being transported to a different time, writing toward the word "unspeakable," and how we turn to poetry in times of great tragedy or celebration.
Tell us about yourself.
I live on the tip of the Saanich peninsula, north of Sidney on Vancouver Island, in a kind of cocoon surrounded by fir trees, where we have to look straight up to see what the weather is on a given day. Hawks, owls and a pair of buck deer also live here and the bucks often venture out into the open to graze beside our horses. Besides my adult poetry, I've written several books in the children's/young adult genre, though they, too, were written as long poems.
What are you currently working on?
At present, I'm writing poems that use a longer line than I've employed in the past. "Borealis" is one of these poems. I find it so interesting how the length of a single line shapes and influences everything else in the poem. Also, I've been thinking of the paintings of Andrew Wyeth, in whose work the appearance of perfect clarity is set against a mysterious, ambiguous quality which creates tension and is exactly what I'm trying for in these poems.
What inspired you to write this poem?
Well, you know how you can be doing some ordinary task, like scrubbing carrots at the sink, and suddenly feel like you're in a wholly different moment of your life, say, twenty years earlier—different landscape, different house. This kind of thing happens to me a lot, and one day I felt taken back to the era when the kids were very small and I was often alone with them on our ranch on the windy side of the Rockies, when our infant daughter fell ill as a blizzard was blowing in. At the time I had that memory, I'd been reading a poem by Brigit Pegeen Kelly that ended with the line, "Something completely understood. But unspeakable." And I wondered how she managed to use such a loaded word as 'unspeakable' so seamlessly, and if I could do it too. So I held onto that memory and wrote toward the word 'unspeakable.'
How long did you work on the poem? How many drafts did you write?
I worked on the poem for a couple of months. After I had a draft on the computer, I'd put the poem up on my screen every day, changing things until I was satisfied. One day I'd think I was finished with the poem, and the next day I'd reread the poem and find another problem to solve. The process continued for weeks.
What do you like most about poetry?
I love the way poetry is the pure distillation of seeing, perceiving, and one's lived experience. Despite the prevailing notion that most North Americans won't read poetry because it's considered to be overly demanding of one's time and attention, whenever we are visited by great tragedy or a momentous celebratory event, inevitably someone will come forward to read a poem. I once attended a funeral in which there was no music at all, but among the half-dozen people who stepped up to speak, four had brought a poem to read. It's the one thing that will suffice when we're standing on the precipice looking at what's happened to us and wondering what on earth there is to say.
When did you know you wanted to be a writer?
Oh, I was very young. I loved hearing the rhythms and rhymes of poems read aloud before my bedtime, and I remember how my mother used to check out a long-play record from the public library of Robert Frost reading his poems. She played it so often I had most of the poems memorized and would recite them to myself as I walked to school. Somewhere in those early years was the seed of this passion for poetry—that I wanted not only the elegance and power of the poems of others around me, I wanted to learn how to make them myself.
What other poets inspire you?
Canadian poets Lorna Crozier and Patrick Lane certainly; American poets Li-Young Lee and Deborah Digges; the Brazillian poet Adelia Prado; the Vietnamese poet Nguyen Quang Thieu, and many others. It's been decades since I drifted off to sleep hearing the lilting rhymes of children's poems, but now when I read I still pay special attention to the poet's use of cadence; if it's one that catches my ear, I'm off and writing.
Pamela Porter's third poetry collection, Cathedral, was shortlisted for the 2011 Pat Lowther Award, and her fourth collection, No Ordinary Place, was shortlisted for the 2013 Raymond Souster Award. Her poems have won the Malahat Review 50th Anniversary Poetry Prize, the Prism International Grand Prize in Poetry, and most recently, the inaugural Gwendolyn MacEwan Best Poem Prize. Her novel in verse, The Crazy Man, won the 2005 Governor General's Award for children's literature. Pamela lives near Sidney, BC, with her family and a menagerie of rescued horses, dogs, and cats, including a formerly wild mustang.


text:
The Canon EF 70-300mm f/4-5.6 IS USM Lens was designed by Canon as a replacement for one of the most popular consumer-grade lenses on the market—the Canon EF 75-300mm f/4.5-5.6 IS. The EF 70-300mm f/4-5.6 touts a compact size, light weight, middle-range price and image stabilization.
The EF 70-300 essentially borrowed the innovative features that made the older Canon 75-300mm one of the most popular lenses on the market and improved upon them. In addition to Canon 3rd generation image stabilization (a feature that allows users to keep images sharp while hand-holding the camera two to three stops lower than normal) the 70-300mm lens includes updated autofocus accuracy, and completely restructured optics.
Both Canon's own comparisons and the observations of many amateur and professional observers note that the Canon 70-300mm performs significantly better than the older 75-300mm IS lens. Additionally, the improved optics allows the lens to be significantly shorter and easier to hand hold. Overall, the Canon EF 70-300mm f/4-5/6 represents an excellent mid-grade offering that improves upon older Canon technology and provides photographers with fast focusing, excellent optics and image stabilization for tripod free shooting in low light conditions.
For photographers who do not require very high image quality at longer focal lengths, fast autofocus and updated image stabilization, the older 75-300mm Canon lens may provide a cheaper alternative to the 70-300mm.
Canon EF 70-300mm Features
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A few photographers, when comparing the 70-300mm IS lens to the older 75-300mm lens, decide that the improvements made to the 70-300mm are unnecessary for their applications and the price premium for the new lens is not worth it.


text:
You might already know Gail Laughton from the inclusion of "Pompeii 76 A.D." in the canonical I Am The Center compilation, or from the same track's inclusion in the Blade Runner score. Alternately, if you're big on 1940's rom-com, you may have heard Laughton's harp recording pantomimed by Cary Grant in The Bishop's Wife—Laughton also instructed Grant in harp-syncing and apparently served as a body double for some close-up shots. Though Laughton worked in Hollywood and played on many cartoon and film soundtracks—John Wayne, Looney Tunes, etc.—Harps of the Ancient Temple was his only solo release, and a radical conceptual departure from his typical work.
Harps uses ancient sacred rituals, each catalogued by year and location, as jumping-off points for his neo-classical and heavily impressionist-influenced interpretations. "Japan 375 A.D.," for example, seems to mimic a koto played in a Japanese pentatonic scale. Much of the record is exactly as pillowy and perfumey as you might hope for from a harp record that's regularly slung around by new age devotees. Still, many tracks lean into dissonance and spin out ominously, building to what feels like like an unobstructed fall down a very long spiral staircase in the closing track, "Atlantis 21,000 B.C." Lots going on here, but happily this works well for both active and passive listening. Fans of Joel Andrews will appreciate this, and similarly it's cloaked in a dense hiss of room tone.


text:
Can anyone advise me on my bumper issue? Whilst I was sat at the roundabout on Sunday and transit van went into the back of my car. Initial damage was minimal. It wasnt til I got home that I realised that although there was no apparent damage to the rear of my car I did notice when I got home that the side parts of the metal work attached to the bumper now is out of alignment to the rest of the bodywork.
Does anyone know how much work is involved with taking the rear bumper off to inspect the possible damage behind the bumper itself? The Transit van was at the standstill point before hitting me and when he did hit my bumper it was square on .
I was pondering trying to repair the damage myself, so that the repair cost would be minimal for the chap that hit me?
I did notice that there are bolts on the inside of the rear wheel arch, which presumable hold the side bit on?


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