for confirmation. Reverse transcription The primary essential parts for this phase include detailing the reaction conditions in full, giving both the amount of RNA used and the total volume of the reaction, give information on the oligonucleotide used as a primer and its concentration, the concentration and type of reverse transcriptase used, and lastly the temperature and amount of time done for the reaction. It is also desirable to have the catalog numbers of reagents used and their manufacturers, the standard deviation for the Cq with and without the transcriptase being involved, and how the cDNA was stored. qPCR target information All of the basic information regarding the target is necessary here, including the gene symbol, the accession database number for the sequence in question, the length of the sequence being amplified, information about the specificity screen used such as BLAST, what splicing variants exist for the sequence, and where the exon or intron for each primer is. There are several desired, but not required information pieces for this section, such as the location of the amplicon, whether any pseudogenes or homologs exist, whether a sequence alignment was done and the data obtained from it, and any data on the secondary structure of the amplified sequence. qPCR oligonucleotides Creation of the oligonucleotides requires only two pieces of essential information: the primer sequences used and the location and details of any modifications made to the sequence. But there are several desirable pieces of data, including the identification number from the RTPrimerDB database, the sequences from the probes, the manufacturer used to make the oligos, and how they were purified. qPCR protocol As one of the primary segments of the guidelines, there are several essential parts on the checklist for the qPCR process itself. This includes the full set of conditions used for the reaction, the volume of both the reaction and the cDNA, the concentrations for the probes, magnesium ions, and dNTPs, what kind of polymerase was used and its concentration, what kit was used and its manufacturer, what additives to the reaction were used, who manufactured the qPCR machine, and what parameters were set for the thermocycling process. The only additional desired pieces of information are the chemical composition of the buffer used, who manufactured the plates and tubes used and what their catalog number is, and whether the reaction was set up manually or by a machine. qPCR validation