Standardizing Q-PCR and developing and testing a new DNA-FISH method for high-throughput assessment of telomere length using genomic DNA

摘要 随着端粒生物学在生物医学和流行病学研究中的日益增长的兴趣， 具有高精度和准确度端粒长度是至关重要的。目前，端粒长度是 在许多不同的实验室使用不同的方法测量，没有标准化。不同 测量和报告端粒长度数据的方法阻碍了跨研究的数据比较和汇集。 对于该领域最常用的端粒长度测量方法的标准化是迫切的 needed.定量PCR（Q-PCR）是目前流行病学最常用的方法， 用于测量平均端粒长度的群体研究。因此，我们建议验证和 标准化Q-PCR程序的关键组成部分，包括PCR前和PCR相关因素， 可重复的端粒长度测量。 尽管Q-PCR端粒长度测量方法具有低成本和高通量的性质， 该方法（和其它现存方法）仅估计平均端粒长度。平均端粒长度为 不能完全提供细胞中端粒长度构成的信息，因为二倍体人类细胞含有92个端粒， 染色体末端和92个染色体末端的端粒长度是高度异质的。高 一种分辨率高、灵敏度高的方法可以测量单个端粒的长度， 对端粒构成的多个方面提供更详细的评估，而不仅仅是平均端粒 长度目的3：建立高通量的DNA荧光原位杂交技术（DNA-FISH） 使用基因组DNA测量单个端粒绝对长度的方法。拟议的新 方法将产生4个端粒参数：1）平均端粒长度; 2）端粒长度变异，定义 作为所有测量的端粒之间的平均端粒长度的变化系数; 3）短端粒的频率 端粒;和4）长端粒的频率。该方法将为研究 环境暴露和心理社会压力不仅影响平均端粒长度的变化， 以及在衰老和疾病易感性的背景下端粒长度变化和短端粒的频率。 本申请是对RFA-AG-19-023“端粒作为环境暴露的哨兵， 心理社会压力与疾病易感性：一项方法比较研究（U 01）。因此，我们建议 以下三个目标：（1）进行合作方法比较研究;（2）合作 制定端粒长度研究的最佳实践指南;（3）开发和验证高通量 使用基因组DNA评估端粒长度构成的方法。

ABSTRACT With a growing interest in telomere biology in biomedical and epidemiological research, measurement of telomere length with high precision and accuracy is of paramount importance. Currently, telomere length is measured in many different laboratories using different methods with no standardization. The different approaches to measure and report telomere length data hinder comparisons and pooling of data across studies. Standardization of the most commonly used telomere length measurement method for the field is urgently needed. Quantitative PCR (Q-PCR) is by far the method most commonly adopted by the epidemiological and population studies for the measurement of average telomere length. Therefore, we propose to validate and standardize key components, including pre-PCR and PCR-related factors, of the Q-PCR procedure for a more reproducible telomere length measurement. Despite the low cost and high-throughput nature of the Q-PCR telomere length measurement method, this approach (and other extant methods) only estimates average telomere length. Average telomere length is not fully informative of telomere length constitution in cells, because a diploid human cell contains 92 chromosomal ends and telomere lengths at the 92 chromosomal ends are highly heterogeneous. A high resolution and sensitive method that can measure the length of single telomeres could advance the field by providing a more detailed assessment of multiple aspects of telomere constitution, not just average telomere length. In aim 3, we propose to develop a high-throughput DNA fluorescent in situ hybridization (DNA-FISH) method for the measurement of absolute lengths of single telomeres using genomic DNA. The proposed new method will generate 4 telomere parameters: 1) average telomere length; 2) telomere length variation, defined as co-efficient of variation of mean telomere length among all measured telomeres; 3) frequency of short telomeres; and 4) frequency of long telomeres. This method will provide a new technology to study how environmental exposures and psychosocial stress impact the changes of not only average telomere length but also telomere length variation and frequency of short telomeres in the context of aging and disease susceptibility. This application is in response to RFA-AG-19-023 “Telomeres as sentinels of environmental exposure, psychosocial stress, and disease susceptibility: a methods comparison study (U01). Accordingly, we propose the following three aims: (1) to conduct a collaborative methods comparison study; (2) to collaborate on developing best practice guidelines for telomere length research; (3) to develop and validate a high-throughput method to assess telomere length constitution using genomic DNA.