Abstract
BackgroundReal-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 standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples.Methodology/Principal FindingsIn this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the “comparative Ct method”. Specifically, we used a raw real-time PCR dataset 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/SignificanceThe obviation of referencing to control samples is attractive whenever 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.
Highlights
Real-time relative quantitative polymerase chain reaction has long been a favoured principle for relative quantifications of nucleic acid sequences
Because more than 18 million run-internal mini standard curves (RIMS)-sets could be constructed in this manner ( = (8,694/2)2), we introduced the constraint that each RIMS-set of Endogenous retrovirus 1 (ERV1) and TURPLE1 must encompass Cq-measurements of samples positioned in the LightCycler carrousel
Real-time relative quantitative PCR in clinical settings is hampered by a lack of accurate and precise approaches to estimate the ratio between nucleic acid sequences without reference to a control sample
Summary
Real-time relative quantitative polymerase chain reaction (qPCR) has long been a favoured principle for relative quantifications of nucleic acid sequences. The original amount of the target sequence is calculated from the parameters of the PCR. The basis of these calculations is the classical PCR equation: NCq~N0:ðEz1ÞCq, modified from 1⁄21 ðI:1Þ. 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 standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples
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