Abstract
BackgroundThe real-time quantitative polymerase chain reaction (qPCR) is routinely used for quantification of nucleic acids and is considered the gold standard in the field of relative nucleic acid measurements. The efficiency of the qPCR reaction is one of the most important parameters in data analysis in qPCR experiments. The Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines recommends the calibration curve as the method of choice for estimation of qPCR efficiency. The precision of this method has been reported to be between SD = 0.007 (three replicates) and SD = 0.022 (no replicates).ResultsIn this article, we present a novel approach to the analysis of qPCR data which has been obtained by running a dilution series. Unlike previously developed methods, our method, Pairwise Efficiency, involves a new formula that describes pairwise relationships between data points on separate amplification curves and thus enables extensive statistics. The comparison of Pairwise Efficiency with the calibration curve by Monte Carlo simulation shows the two-folds improvement in the precision of estimations of efficiency and gene expression ratios on the same dataset.ConclusionsThe Pairwise Efficiency nearly doubles the precision in qPCR efficiency determinations compared to standard calibration curve method. This paper demonstrates that applications of combinatorial treatment of data provide the improvement of the determination.
Highlights
The real-time quantitative polymerase chain reaction is routinely used for quantification of nucleic acids and is considered the gold standard in the field of relative nucleic acid measurements
The calibration curve method is widely considered the most precise method for quantitative polymerase chain reaction (qPCR) efficiency estimation [13] and is required in the MIQE guidelines: “Calibration curves for each quantified target must be included with the submitted manuscript, slopes and y intercepts derived from these calibration curves must be included with the publication” [5]
Our results show that the application of Pairwise Efficiency makes it possible to nearly double the precision in qPCR efficiency determinations without increasing the pipetting workload and minimizing cost
Summary
The real-time quantitative polymerase chain reaction (qPCR) is routinely used for quantification of nucleic acids and is considered the gold standard in the field of relative nucleic acid measurements. The Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines recommends the calibration curve as the method of choice for estimation of qPCR efficiency. The precision of this method has been reported to be between SD = 0.007 (three replicates) and SD = 0.022 (no replicates). Real-time qPCR is considered the most sensitive technique for nucleic acid quantification, and enables measurements on as few as several molecules of the target [1] The advantage of this method over earlier methods of quantification, such as end-point PCR followed by gel visualization, is the ability to account for the efficiency of the PCR reaction by following it in real time and gathering fluorescence data after each amplification cycle [2,3,4]. The downside of this approach is the increased workload and cost
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