Increasing the accuracy and precision of relative telomere length estimates by RT qPCR
Increasing the accuracy and precision of relative telomere length estimates by RT qPCR
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DOI：
10.1111/1755-0998.12711
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发表时间：
2018-01-01
影响因子：
7.7
通讯作者：
Peters, Anne
中科院分区：
文献类型：
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作者：
Eastwood, Justin R.;Mulder, Ellis;Peters, Anne
由于保护染色体完整性的DNA帽端粒的磨损被各种形式的应激加速，端粒长度（TL）已被提出作为一生累积应激的指标。在生态学研究中，它被用来提供对衰老、生活史权衡、繁殖成本和疾病的见解。qPCR是一种高通量和低成本的相对TL （rTL）测量工具，可用于新采集和存档的生态样品。然而，qPCR容易受到方法本身和分析前步骤的误差。在这里，可重复性在多个层面（测定内、测定间和提取间）进行了全面和单独的评估，以阐明测量误差的原因，作为提高精度的一步。我们还测试了如何提高准确性，定义为TL估计的“金标准”（端粒限制性片段长度分析与凝胶内杂交）之间的相关性。我们发现qPCR的重复性（测定内和测定间水平）在三种常见的储存介质（乙醇、朗迈尔和女王）中处于相似的水平。然而，在Queen’s lysis缓冲液中存储的样品，萃取间的重复性降低了50%，表明存储介质会影响精度。通过估计多个qPCR反应和多次提取的rTL，可以提高精密度和准确度。重复增加的统计能力相当于25%（单次提取分析两次）和17%（两次提取）的样本量增加。总体而言，本研究确定了高通量端粒定量的新变异性来源，并为采样策略设计以及如何提高rTL精度和准确性提供了指导。
As attrition of telomeres, DNA caps that protect chromosome integrity, is accelerated by various forms of stress, telomere length (TL) has been proposed as an indicator of lifetime accumulated stress. In ecological studies, it has been used to provide insights into ageing, life history trade-offs, the costs of reproduction and disease. qPCR is a high-throughput and cost-effective tool to measure relative TL (rTL) that can be applied to newly collected and archived ecological samples. However, qPCR is susceptible to error both from the method itself and pre-analytical steps. Here, repeatability was assessed overall and separately across multiple levels (intra-assay, inter-assay and inter-extraction) to elucidate the causes of measurement error, as a step towards improving precision. We also tested how accuracy, defined as the correlation between the "gold standard" for TL estimation (telomere restriction fragment length analysis with in-gel hybridization), could be improved. We find qPCR repeatability (intra- and inter-assay levels) to be at similar levels across three common storage media (ethanol, Longmire's and Queen's). However, inter-extraction repeatability was 50% lower for samples stored in Queen's lysis buffer, indicating storage medium can influence precision. Precision as well as accuracy could be increased by estimating rTL from multiple qPCR reactions and from multiple extractions. Repetition increased statistical power equivalent to a 25% (single extraction analysed twice) and 17% (two extractions) increase in sample size. Overall, this study identifies novel sources of variability in high-throughput telomere quantification and provides guidance on sampling strategy design and how to increase rTL precision and accuracy.