Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94158, United States.
Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94158, United States.
Psychoneuroendocrinology. 2019 Jan;99:271-278. doi: 10.1016/j.psyneuen.2018.10.005. Epub 2018 Oct 10.
Research in the last decade has explored the length of telomeres, the protective ends of eukaryotic chromosomes, as a biomarker for the cumulative effects of environmental exposures and life experiences as well as a risk factor for major diseases. With a growing interest in telomere biology across biomedical, epidemiological and public health research, it is critical to ensure that the measurement of telomere length is performed with high precision and accuracy. Of the several major methods utilized to determine telomere length, quantitative PCR (qPCR) remains the most cost-effective and suitable method for large-scale epidemiological and population studies. However, inconsistencies in recent reports utilizing the qPCR method highlight the need for a careful methodological analysis of each step of this process. In this review, we summarize each critical step in qPCR telomere length assay, including sample type selection, sample collection, storage, processing issues and assay procedures. We provide guidance and recommendations for each step based on current knowledge. It is clear that a collaborative and rigorous effort is needed to characterize and resolve existing issues related to sample storage, both before and after DNA extraction, as well as the impact of different extraction protocols, reagents and post extraction processing across all tissue types (e.g. blood, saliva, buccal swabs, etc.) to provide the needed data upon which best practices for TL analyses can be agreed upon. Additionally, we suggest that the whole telomere research community be invited to collaborate on the development and implementation of standardized protocols for the assay itself as well as for reporting in scientific journals. The existing evidence provides substantial support for the continuation of telomere research across a range of different exposures and health outcomes. However, as with any technological or methodologic advance in science, reproducibility, reliability and rigor need to be established to ensure the highest quality research.
在过去的十年中，研究人员探索了端粒的长度，即真核染色体的保护末端，将其作为环境暴露和生活经历的累积效应以及主要疾病风险因素的生物标志物。随着人们对端粒生物学在生物医学、流行病学和公共卫生研究中的兴趣日益浓厚，确保端粒长度的测量具有高精度和准确性至关重要。在用于确定端粒长度的几种主要方法中，定量聚合酶链反应（qPCR）仍然是大规模流行病学和人群研究中最具成本效益和适用性的方法。然而，最近利用 qPCR 方法的报告中的不一致性突出表明，需要仔细分析该过程的每个步骤的方法学。在这篇综述中，我们总结了 qPCR 端粒长度分析的每个关键步骤，包括样本类型选择、样本采集、储存、处理问题和分析程序。我们根据当前的知识为每个步骤提供了指导和建议。显然，需要协作和严格的努力来描述和解决与样本储存相关的现有问题，包括在 DNA 提取之前和之后，以及不同提取方案、试剂和提取后处理对所有组织类型（例如血液、唾液、口腔拭子等）的影响，以提供有关 TL 分析的最佳实践可以达成一致的必要数据。此外，我们建议邀请整个端粒研究界合作制定和实施标准化协议，用于该分析本身以及科学期刊中的报告。现有证据为在一系列不同的暴露和健康结果中继续进行端粒研究提供了充分的支持。然而，就像科学中的任何技术或方法进步一样，需要建立可重复性、可靠性和严谨性，以确保最高质量的研究。
