How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.
How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.
复制标题
DOI：
10.1016/j.bdq.2015.01.005
复制
发表时间：
2015-03
影响因子：
--
通讯作者：
Kubista M
中科院分区：
文献类型：
--
作者：
Svec D;Tichopad A;Novosadova V;Pfaffl MW;Kubista M
在大量技术重复的策略性试验设计的基础上，我们用标准曲线检验了用标准曲线估计PCR效率的不精确性。特别是，就一个常见的不同因素而言，这一估计的稳健性如何：所使用的仪器、进行的技术重复次数以及在整个稀释系列中转移的体积的影响。我们使用了6种不同的定量聚合酶链式反应仪器，每个浓度进行1-16次定量聚合酶链式反应，我们分别测试了2-10μL转移的分析物体积。我们发现，不同的仪器估计的聚合酶链式反应效率有很大差异。使用蒙特卡罗方法，我们发现，如果在16个不同的平板上使用只有一个qPCR重复的标准曲线，那么PCR效率估计的不确定度可能高达42.5%(95%CI)。基于我们的研究，我们提出了精确估计PCR效率的建议：(1)应生成一条稳健的标准曲线，每个浓度至少有3-4个qPCR重复；(2)效率依赖于仪器，但在一个平台上可重复性稳定；(3)在构建连续稀释系列时使用更大的体积，可以减少采样误差，并能够在更大的动态范围内进行校准。
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.