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




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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.



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

Fuente: http://plos.srce.hr/



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