A reverse transcription (RT) reaction containing 1 ^g total RNA should yield enough cDNA for at least 20 genes to be examined. Based upon a total RNA yield from whole eye of 10 ^g, this should provide enough material for 200 transcripts to be examined. In contrast, a retinal extraction yielding 3 ^g will enable 60 transcripts to be profiled.
When approaching the analysis of multiple transcripts in this manner, the inclusion of an IC on every qPCR run is unnecessary, and simply consumes reagents and space on a plate. As comparisons for a given transcript are made relative to each other, normalization to ICs run on different plates on different days is valid. One way of illustrating this point is to divide the expression of a target gene to the mean target gene expression, so that target gene expression is corrected relative to the mean of all samples. Second, carry out the same procedure for an IC measured for the same samples, correcting to the mean IC expression. If the mean-corrected target gene expression is now normalized to the mean-corrected IC expression, the relative fold change and variance observed between any group of samples will be preserved. This offers a great advantage for relative quantification, as it means that multiple ICs can be used based upon different experimental runs, and barring quite dramatic freeze-thaw effects, which may be evaluated by differences in IC profiles, a target gene run one day may be normalized to a IC run days or even weeks earlier (or later). For this reason, any samples between which comparisons are to be made should be included within the same plate to ensure that they are truly comparable.
Once ICs are well-characterized within a group of cDNA samples, and a normalization factor (NF) is calculated for each sample, this value can be used to normalize all subsequent target genes. Given the synthesis of 20 ^l of cDNA, and using 1 ^l per target gene, a researcher would be wise to first analyze the expression of two or three (or even more) ICs to ensure that an accurate NF can be calculated before moving on to investigate up to 16 or 17 target genes. However, if a second RT is performed on the same RNA, re-deriving the NF is necessary, as differences in the RT efficiency may occur between the first and second batches of cDNA, and the same relationship between target gene and IC expression may not hold in the second batch of cDNA. This raises an unresolved issue with one-step RT-PCR protocols, where a different reverse transcription is conducted for every reaction.
The use of kinetic approaches to qPCR enables researchers to improve assay throughput, removing the need for using space on each plate for standard curves (which may even demonstrate different reaction kinetics to the cDNA samples under analysis). The use of kinetic data already collected during the course of a qPCR to determine E, coupled with a two-step procedure of cDNA synthesis with subsequent NF derivation enables researchers to move from candidate gene to expression profile in a single reaction, greatly improving assay throughput and saving both time and resources.
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