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

摘要 随着人们对生物医学和流行病学研究中的端粒生物学越来越感兴趣，端粒生物学的测量 具有高精确度和准确度的端粒长度至关重要。目前，端粒长度为 在许多不同的实验室使用不同的方法进行测量，没有标准化。不同的 测量和报告端粒长度数据的方法阻碍了跨研究数据的比较和汇集。 现场最常用的端粒长度测量方法的标准化是当务之急 需要的。定量聚合酶链式反应(Q-聚合酶链式反应)是迄今为止流行病学和 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，包括预聚合酶链式反应和聚合酶链式反应相关因素，以获得更多 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特性， 这种方法(和其他现存的方法)只估计平均端粒长度。平均端粒长度为 对细胞中端粒长度的构成没有充分的信息，因为一个二倍体人类细胞含有92 92条染色体末端的染色体末端和端粒长度具有高度的异质性。一次高潮 分辨率和灵敏的方法可以测量单个端粒的长度，可以通过 提供对端粒构成的多个方面的更详细的评估，而不仅仅是普通的端粒 长度。在目标3中，我们建议建立一种高通量的dna荧光原位杂交技术(dna-Fish)。 利用基因组DNA测量单个端粒绝对长度的方法。建议的新 方法将生成4个端粒参数：1)平均端粒长度；2)端粒长度变化，定义 作为所有测量的端粒之间平均端粒长度变化的系数；3)短的频率 端粒；4)长端粒出现的频率。这种方法将提供一种新技术来研究 环境暴露和心理社会应激不仅影响端粒平均长度的变化，而且影响端粒长度的变化 此外，在衰老和疾病易感性的背景下，端粒长度的变化和短端粒的频率也是如此。 本申请是对作为环境暴露的前哨的RFA-AG-19-023“端粒的响应， 心理社会压力与疾病易感性：一项方法比较研究(U01)。因此，我们建议 以下三个目标：(1)进行协作方法比较研究；(2)协作 制定端粒长度研究的最佳实践指南；(3)开发和验证高通量 方法利用基因组DNA评价端粒长度构成。

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.