PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency

Abstract

This study addresses the problem of PCR inhibition by reverse transcriptase. It has been shown that the inhibition occurs mostly when a small amount of RNA is taken for RT reaction, and it is more visible for rarely expressed transcripts. We show here that the inhibition takes place regardless of what amount of template is utilized for RT. The inhibition possesses a global nature, i.e. the amplification of any given transcript may be compromised with different levels of inhibition. The process of inhibition also explains wrongfully derived PCR amplification efficiencies, sometimes more than 100%, when the sequential dilutions of unpurified RT sample are utilized to build the calibration curve. The RT influences PCR not only by inhibiting it. When microgram(s) of RNA are taken for RT reaction, reverse transcriptase may cause overamplification of some transcripts under certain PCR conditions. The possible mechanism of RT influence on PCR is presented, and a purification method is implemented to remove the effects of RT on PCR.

Figure 1

Experimental layout: comparison of unpurified and PCI samples.

Figure 2

Experiments to detect DNA synthesis activity of RT.

Figure 3

The PCI procedure improves the real-time PCR results, when 50 ng of total RNA was taken for RT reaction. The dilutions correspond to certain volumes of undiluted RT reaction—1.28, 0.64, 0.16, 0.04 and 0.01 µl were used as a template in PCR with a B2M pair of primers (A) and a TBP pair ofprimers (B).