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

标准化 Q-PCR 并开发和测试新的 DNA-FISH 方法，以使用基因组 DNA 高通量评估端粒长度

• 批准号: 10179395
• 负责人: YUN-LING ZHENG
• 金额: $ 31.1万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-12 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10179395
• 关键词: Adopted; Affect; Aging; Biology; Biomedical Research; Breast Cancer Risk Factor; Cells; Chromosomes; Constitution; DNA; Data; Data Pooling; Detection; Development; Diploidy; Disease; Disease susceptibility; Educational workshop; Environmental Exposure; Fluorescent in Situ Hybridization; Frequencies; Future; Genes; Genomic DNA; Goals; Human; Individual; Investigation; Knowledge; Laboratories; Length; Malignant neoplasm of lung; Malignant neoplasm of urinary bladder; Measurement; Measures; Metaphase; Methods; Nature; Pathway interactions; Population Study; Practice Guidelines; Procedures; Process; Psychosocial Stress; Public Health; Recommendation; Reporting; Reproducibility; Reproducibility of Results; Research; Research Design; Resolution; Resources; Role; Sampling; Sentinel; Standardization; Statistical Data Interpretation; Testing; Tissues; United States National Institutes of Health; Variant; Work; cancer risk; cell type; cost; epidemiology study; experience; human disease; improved; interest; new technology; novel; response; risk prediction; sample collection; telomere; tool

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.

抽象的 随着生物医学和流行病学研究中对端粒生物学的兴趣日益浓厚， 高精度和准确的端粒长度至关重要。目前，端粒长度是最重要的。 许多不同的实验室使用不同的方法进行测量，没有标准化。 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。 该领域最常用的端粒长度测量方法的标准化刻不容缓 定量PCR（Q-PCR）是迄今为止流行病学和流行病学最常用的方法。 因此，我们建议进行验证和测量平均端粒长度。 对 Q-PCR 程序的关键组成部分（包括 PCR 前和 PCR 相关因素）进行标准化，以实现更准确的结果 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点， 这种方法（和其他现有方法）仅估计平均端粒长度。 不能完全了解细胞中端粒长度的构成，因为二倍体人类细胞包含 92 92条染色体末端的染色体末端和端粒长度具有高度异质性。 能够测量单个端粒长度的分辨率和灵敏方法可以通过以下方式推进该领域的发展： 提供对端粒构成多个方面的更详细评估，而不仅仅是平均端粒 在目标 3 中，我们建议开发高通量 DNA 荧光原位杂交 (DNA-FISH)。 提出的使用基因组 DNA 测量单个端粒绝对长度的新方法。 方法将生成 4 个端粒参数：1) 平均端粒长度；2) 端粒长度变异，已定义 作为所有测量端粒之间平均端粒长度的变异系数 3) 短频率； 4）长端粒的频率该方法将为研究如何进行提供新技术。 环境暴露和社会心理压力不仅影响平均端粒长度的变化，而且影响端粒长度的变化。 还有衰老和疾病易感性背景下的端粒长度变化和短端粒频率。 此申请是为了响应 RFA-AG-19-023“端粒作为环境暴露的哨兵， 心理社会压力和疾病易感性：一项方法比较研究（U01）。 以下三个目标：（1）进行协作方法比较研究；（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。