Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a approximate to 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P < 0.001, n = 4), and a approximate to 4-fold increase in copy number with a specific hexamer (P < 0.001, n = 4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.