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Questioning comparative quantitative polymerase chain reaction (qPCR) assumptions
Comparative quantification is now almost a routine method for analyzing quantitative polymerase chain reaction (qPCR) data. This method employs reference transcripts to minimize sample variability, but there is little experimental evidence to support that all transcripts behave identically within the same system. Reference transcripts could introduce experimental bias if there is any difference in reference and target transcripts degradation rates or RT efficiency. Since endogenous reference transcripts have a great influence on comparative qPCR studies, the overall aim of this study was to examine the sensitivity of eleven endogenous reference transcripts to RNA degradation. The experimental results demonstrate that heating RNA at 37°C, 70°C and 90°C decreased RNA integrity resulting in a first order RNA degradation process, in which degradation rates stratified into two groups with different half-lives (short versus long). The implications of the disparity in transcript half-life are apparent when using an endogenous reference transcript to control for RNA integrity. The results suggest when both the reference and target transcripts have similar half-lives, the original transcript distribution can be maintained even when RNA integrity is comprised, but cannot be maintained when both these transcripts have significantly different half-lives. The halflives of transcripts seem to correlate with GC content and number of exons per transcript. In conclusion, these studies suggest that reference transcripts should be selected so that they have similar half-lives to the target transcript to control for variability in RNA integrity.