Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry.
Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry.
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DOI：
10.1186/1471-2105-9-221
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发表时间：
2008-04-29
影响因子：
3
通讯作者：
Ritz C
中科院分区：
文献类型：
--
作者：
Spiess AN;Feig C;Ritz C
四参数Sigmoid模型是定量实时聚合酶链式反应(QPCR)数据分析中常用的方法之一。我们观察到，由于拐点周围对称性的内在限制，这些模型在拟合结果上并不是最优的。因此，我们发现有必要采用一种数学算法来绕过这个问题，并利用一个额外的参数来适应S型qPCR数据中的不对称结构。四参数模型与五参数模型通过基于残差方差的嵌套F检验进行了比较，从而获得了更高性能的统计度量。对于我们检查的几乎所有的qPCR数据，五参数模型得到了明显更好的拟合。此外，使用四个独立的稀释数据集评估了效率估计和定量比率计算的准确性和精确度，并与最常用的量化方法进行了比较。结果表明，五参数模型比非S型量化方法具有更高的准确度和精密度。五参数S型模型的预测效果优于已建立的四参数模型，具有较高的统计意义。对重要的聚合酶链式反应参数，如聚合酶链式反应效率、阈值循环数和初始模板荧光的估计更稳健，并且具有较小的方差。该模型在可自由获得的统计R环境下的qPCR程序包中实现。该程序包可从作者的主页下载。
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. 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. 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.
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影响因子：
2.8
作者：
Pfaffl, MW;Lange, IG;Meyer, HHD
通讯作者：
Meyer, HHD
影响因子：
2.9
作者：
Gottschalk, PG;Dunn, JR
通讯作者：
Dunn, JR
影响因子：
1.7
作者：
Zhao, S;Fernald, RD
通讯作者：
Fernald, RD
影响因子：
14.9
作者：
Tichopad, A;Dilger, M;Pfaffl, MW
通讯作者：
Pfaffl, MW
DOI：
10.1016/s1056-8719(99)00026-x
发表时间：
1999-04-01
影响因子：
1.9
作者：
Van der Graaf, PH;Schoemaker, RC
通讯作者：
Schoemaker, RC