Consequences of measurement error in qPCR telomere data: A simulation study
Consequences of measurement error in qPCR telomere data: A simulation study
复制标题
DOI：
10.1371/journal.pone.0216118
复制
发表时间：
2019-05-01
期刊：
影响因子：
3.7
通讯作者：
Bateson, Melissa
中科院分区：
文献类型：
--
作者：
Nettle, Daniel;Seeker, Luise;Bateson, Melissa
定量聚合酶链式反应方法提供了一种廉价、快速的方法来估计一组生物样本的相对端粒长度。像所有的实验室方法一样，它包含一定程度的测量误差。相对端粒长度的估计是通过对实际测量(端粒和对照基因的CQ值)进行非线性转换并将它们组合成一个比率(TS比率)来完成的。在这里，我们使用计算机模拟，并辅之以数学分析，来探索在横截面和纵向数据中，测量误差如何影响相对端粒长度的qPCR估计。我们发现，当计算TS比时，在CQ值水平上引入的误差被放大。如果CQ水平的误差是正态分布的，并且与真实端粒长度无关，那么TS比率中的误差是正偏斜的，并且与真实端粒长度成正比。随着端粒和/或对照基因CQ值测量误差的增加，TS比值的重复性急剧下降。在模拟的纵向数据中，仅测量误差就可以在相对端粒长度的连续测量之间产生低相关性的模式，并且与初始相对端粒长度的变化率具有强烈的负相关性。我们的结果说明了减少测量误差的重要性：CQ值误差的微小增加可以对相对端粒长度的qPCR估计的能力和可解释性产生重大影响。这些发现还说明了在每个数据集中表征测量误差的重要性--变异系数通常无济于事，研究人员应该报告CQ值的标准偏差和/或TS比率的重复性--并在解释TS比率随时间变化的模式时考虑测量误差的已知影响。
The qPCR method provides an inexpensive, rapid method for estimating relative telomere length across a set of biological samples. Like all laboratory methods, it involves some degree of measurement error. The estimation of relative telomere length is done subjecting the actual measurements made (the Cq values for telomere and a control gene) to non-linear transformations and combining them into a ratio (the TS ratio). Here, we use computer simulations, supported by mathematical analysis, to explore how errors in measurement affect qPCR estimates of relative telomere length, both in cross-sectional and longitudinal data. We show that errors introduced at the level of Cq values are magnified when the TS ratio is calculated. If the errors at the Cq level are normally distributed and independent of true telomere length, those in the TS ratio are positively skewed and proportional to true telomere length. The repeatability of the TS ratio declines sharply with increasing error in measurement of the Cq values for telomere and/or control gene. In simulated longitudinal data, measurement error alone can produce a pattern of low correlation between successive measures of relative telomere length, coupled with a strong negative dependency of the rate of change on initial relative telomere length. Our results illustrate the importance of reducing measurement error: a small increase in error in Cq values can have large consequences for the power and interpretability of qPCR estimates of relative telomere length. The findings also illustrate the importance of characterising the measurement error in each dataset-coefficients of variation are generally unhelpful, and researchers should report standard deviations of Cq values and/or repeatabilities of TS ratios-and allowing for the known effects of measurement error when interpreting patterns of TS ratio change over time.