Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry.
BACKGROUND
Fitting four-parameter sigmoidal models is one of the methods established in the analysis of quantitative real-time PCR (qPCR) data. We had observed that these models are not optimal in the fitting outcome due to the inherent constraint of symmetry around the point of inflection. Thus, we found it necessary to employ a mathematical algorithm that circumvents this problem and which utilizes an additional parameter for accommodating asymmetrical structures in sigmoidal qPCR data.


RESULTS
The four-parameter models were compared to their five-parameter counterparts by means of nested F-tests based on the residual variance, thus acquiring a statistical measure for higher performance. For nearly all qPCR data we examined, five-parameter models resulted in a significantly better fit. Furthermore, accuracy and precision for the estimation of efficiencies and calculation of quantitative ratios were assessed with four independent dilution datasets and compared to the most commonly used quantification methods. It could be shown that the five-parameter model exhibits an accuracy and precision more similar to the non-sigmoidal quantification methods.


CONCLUSIONS
The five-parameter sigmoidal models outperform the established four-parameter model with high statistical significance. The estimation of essential PCR parameters such as PCR efficiency, threshold cycles and initial template fluorescence is more robust and has smaller variance. The model is implemented in the qpcR package for the freely available statistical R environment. The package can be downloaded from the author's homepage.
Spiess
Andrej-Nikolai
AN
Department of Andrology, University Hospital Hamburg-Eppendorf, Hamburg, Germany. a.spiess@uke.uni-hamburg.de
Feig
Caroline
C
Ritz
Christian
C
D000465:Algorithms; D009716:Numerical Analysis, Computer-Assisted; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*