Joglekar Mugdha V, Satoor Sarang N, Wong Wilson K M, Cheng Feifei, Ma Ronald C W, Hardikar Anandwardhan A
Diabetes and Islet biology, NHMRC Clinical Trials Centre, Faculty of Medicine and Health, University of Sydney, Camperdown, NSW 2150, Australia.
DNA Sequencing Laboratory, National Centre for Cell Science, NCMR Campus, Sai Trinity Complex, Pashan, Pune 411 021, India.
Methods Protoc. 2020 Apr 3;3(2):27. doi: 10.3390/mps3020027.
Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease.
端粒代表染色体末端的核苷酸重复序列，对染色体稳定性至关重要。它们在每一轮DNA复制时都会缩短，主要原因是滞后链的DNA合成不完全。相对端粒长度的缩短与衰老和一系列疾病状态相关。有多种方法可用于测量端粒长度，如末端限制片段分析、实时定量PCR（qPCR）和荧光原位杂交；然而，基于qPCR的方法通常用于大规模人群研究。目前报道的基于qPCR的方法有多种变体，包括单拷贝基因的选择、引物序列、试剂以及不同研究中的数据分析方法。在此，我们提供了一个详细的分步方案，该方案我们已经进行了优化，并在其他用户手中成功测试。该方案将帮助有兴趣测量细胞或更大临床队列/研究样本中端粒相对长度的研究人员确定端粒长度与健康和疾病的关联。
