Article

Amplification Efficiency: Linking Baseline and Bias in the Analysis Of Quantitative PCR Data

Heart Failure Research Center, Academic Medical Center, University of Amsterdam, The Netherlands.
Nucleic Acids Research (Impact Factor: 9.11). 03/2009; 37(6):e45. DOI: 10.1093/nar/gkp045
Source: PubMed

ABSTRACT

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data
are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence
baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values
and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because
of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of
the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing
the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible
PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points
in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of
these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

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