Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods
Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods
复制标题
DOI：
10.1186/1471-2156-13-77
复制
发表时间：
2012-09-06
期刊：
影响因子：
2.9
通讯作者：
Palsboll, Per J.
中科院分区：
文献类型：
--
作者：
Olsen, Morten Tange;Berube, Martine;Palsboll, Per J.
背景：端粒是染色体的保护帽，在模式物种和非模式物种中已成为生物年龄和生活史的强大标记。用于端粒长度估计的qPCR方法是最常见的端粒长度估计方法之一，但最近受到了批评，因为它太容易出错，结果不可靠。这一批评恰逢人们对定量聚合酶链式反应技术的潜力和局限性的认识不断增加，以及定量聚合酶链式反应实验、分析和报告步骤标准化的通用指南(MIQE)的提出。为了评估qPCR方法在非模式物种端粒长度估计中的实用性，我们针对座头鲸端粒进行了四种不同的qPCR方法，并随后进行了严格的质量控制以评估每种方法的性能。结果：不同方法的性能有很大差异，只有一种方法对座头鲸的端粒长度估计有效。造成这些分析间差异的最显著因素是引物设计和使用单链或多链分析的选择。根据检测和定量方法的不同，推测的扩增效率差异高达40%，然而这种差异只影响表现最差的检测方法中的端粒长度估计。结论：我们的结果表明，表面上表现良好的qPCR检测可能包含只有通过广泛的质量控制才能检测到的偏差。此外，我们还表明，如果致力于在所有实验和分析步骤中进行优化，用于端粒长度估计的qPCR方法可以具有很高的精确度和准确性，因此适合于非模式物种的端粒测量。最后，我们强调了一组可能有助于进一步标准化端粒长度估计的qPCR方法的质量控制，并讨论了可能导致qPCR实验中差异的一些因素。
Background: Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay.Results: Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays.Conclusion: Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.