Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves

Abstract

Background: Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional tandard curves and provide both run-specific and target-specific estimates of PCR arameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples. & Methodology/Principal Findings: In this study, we ompared RIMS-based drqPCR with classical quantifications based on external standard curves and the ''comparative Ct method''. Specifically, we used a raw real-time PCR ataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy. & Conclusions/Significance: The obviation of referencing to control samples is attractive henever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified. © 2010 Bernth Jensen et al.