Standardized determination of real-time PCR efficiency from a single reaction set-up
Standardized determination of real-time PCR efficiency from a single reaction set-up
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DOI：
10.1093/nar/gng122
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发表时间：
2003-10-15
影响因子：
14.9
通讯作者：
Pfaffl, MW
中科院分区：
文献类型：
--
作者：
Tichopad, A;Dilger, M;Pfaffl, MW
提出了一种实时PCR扩增效率的计算方法。它是基于实时PCR动力学中指数行为观察开始的统计定界。通过单独的数学算法从计算过程中排除PCR基础荧光期、非指数期和平台期。我们在LightCycler平台上获得的多个目标的实验数据上验证了该方法。所开发的方法产生的结果比目前使用的方法的系列稀释扩增效率估计更高的准确性。单反应设置估计对样品中靶序列起始浓度的差异敏感。此外，它抵制了研究人员的主观影响，因此估计可以完全工具化。
We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.