Minimization of PCR efficiency differences between standards and samples through dilution of PCR amplicons in reverse transcription buffer

Abstract

Quantitative real-time PCR (QPCR) [1] is a widespread method for gene expression analysis. This article concerns two-steps, two-enzyme relative QPCR [2–4] using standard curves for determination of raw expression levels An important potential source of biases in the procedure [5,6] is PCR efficiency [7,8]. Because calculation of expression levels relies on the use of standards, it is imperative that both standards and samples have similar PCR efficiencies. This becomes particularly important when normalization is performed with housekeeping genes [9] because biases arising from the target and normalization genes are additive. A number of studies have shown that reverse transcription (RT) enzymes and reaction mixtures may cause adverse effects on PCR efficiency [10–13]. Difference in PCR efficiencies can be minimized through purification of samples after the RT step along with use of serial dilutions of purified complementary DNA (cDNA) as standards. However, factors such as number and availability of samples and target copy abundance make this approach potentially unsuitable. PCR amplicons, positive cDNAs, and plasmid DNAs are broadly used to prepare QPCR standard curves. Depending on experimental needs, these