Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.
Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.
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DOI：
10.1093/nar/gkp045
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发表时间：
2009-04
影响因子：
14.9
通讯作者：
Moorman AF
中科院分区：
文献类型：
--
作者：
Ruijter JM;Ramakers C;Hoogaars WM;Karlen Y;Bakker O;van den Hoff MJ;Moorman AF
尽管定量聚合酶链式反应(QPCR)在定量mRNA转录本中起着核心作用，但对qPCR数据的大多数分析仍然委托给qPCR仪器附带的软件。对于荧光基线的处理尤其如此。这篇文章表明，基线估计误差直接反映在观察到的聚合酶链式反应效率值中，因此在估计的起始浓度和“折叠差”结果中呈指数传播。由于基线荧光的来源和动力学未知，在聚合酶链式反应的初始周期中监测的荧光值不能用于估计有用的基线数值。开发了一种算法，通过从聚合酶链式反应的早期平台阶段向下重建对数线性阶段来估计基线，并显示出非常重复性的聚合酶链式反应效率值。通过将回归线拟合到对数线性阶段中的数据点的子集来确定每个样本的PCR效率值。当在计算每个样本的起始浓度估计时，使用每个扩增子的这些PCR效率的平均值来计算qPCR结果中的变异性以及偏差显著降低。
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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