real-time relative qpcr without reference to control samples and estimation of run-specific pcr parameters from run-internal mini-standard curves实时相对qpcr没有参考控制样品和运行特定pcr参数估计run-internal mini-standard曲线.pdfVIP

Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves 1 1 2¤ 2 Jens Magnus Bernth Jensen *, Mikkel Steen Petersen , Marc Stegger , Lars J. Østergaard , Bjarne K. Møller1 1 Department of Clinical Immunology, University Hospital of Aarhus, Skejby, Denmark, 2 Department of Infectious Diseases, University Hospital of Aarhus, Skejby, Denmark 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 quanti