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Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves

2010; Public Library of Science; Volume: 5; Issue: 7 Linguagem: Inglês

10.1371/journal.pone.0011723

1932-6203

Jens Magnus Bernth Jensen, Mikkel Steen Petersen, Marc Stegger, Lars Østergaard, Bjarne Kuno Møller,

RNA Research and Splicing

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 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 Findings In 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/Significance The 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.