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

Jens Magnus Bernth Jensen, Mikkel Steen Petersen, Marc Stegger, Lars J Østergaard and Bjarne K Møller

PLOS ONE, 2010, vol. 5, issue 7, 1-8

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 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.

Date: 2010
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pone00:0011723

DOI: 10.1371/journal.pone.0011723

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