Development of a novel assay for the analysis of newly synthesized RNA

描述(申请人提供)：虽然信使核糖核酸衰减率的变化约占细胞中基因表达调控的50%，但目前还没有准确和可靠地测量信使核糖核酸半衰期的分析方法。特别是，缺乏可靠的商业试剂盒，使普通生物医学研究实验室能够有效地研究信使核糖核酸的衰减率，大大减缓了这一领域的进展。为了满足这一需求，我们开发了一种新的方法，涉及代谢标记的RNA分子的直接PCR分析来确定mRNA的衰减率。在这一应用中，我们建议证明这种新方法的可行性，并在两个目标上验证其有效性。首先，我们将优化一种独特的核苷类似物代谢标记技术，该技术利用一种简单且经过历史验证的结合化学方法，并选择性地对所需的mRNA群体进行RT-PCR扩增。其次，我们将使用合成的mRNAs和几个特征良好的内源性细胞mRNAs来验证我们的代谢标记技术/RT-PCR方法。总的来说，这套试剂盒将实现一种无偏见、用户友好、可靠的方法，用于常规测定信使核糖核酸半衰期和研究受调控的核糖核酸降解。 与公共卫生相关：目前研究细胞中RNA降解率的方法存在程序性偏见，而且不可靠。虽然自制方法是可用的，但它们通常会浪费实验室人员的时间进行分析优化和验证。鉴于信使核糖核酸衰减率的变化可能是细胞中近一半的基因表达调控的原因，缺乏一种可靠的技术来准确评估信使核糖核酸降解率正在阻碍许多努力。因此，我们提出了一种解决这些问题的新方法。

DESCRIPTION (provided by applicant): While changes in mRNA decay rates account for approximately 50% of the regulation of gene expression in the cell, an assay to accurately and reliably measure mRNA half lives is currently not available. In particular, the deficiency of reliable commercial kits which enable the average biomedical research lab to effectively investigate mRNA decay rates is significantly slowing down progress in this area. In order to meet this need, we have developed a novel approach involving the direct PCR analysis of metabolically labeled RNA molecules to determine mRNA decay rates. In this application, we propose to demonstrate feasibility of this novel approach and validate its effectiveness in two aims. First, we will optimize a unique nucleoside analog metabolic labeling technique which utilizes a simple and historically validated conjugation chemistry approach coupled with selective RT-PCR amplification of the desired mRNA population. Second, we will validate our metabolic labeling technique/RT-PCR approach using synthetic mRNAs and several well-characterized endogenous cellular mRNAs. Collectively this kit will enable a non-biased, user-friendly, reliable method for the routine determination of mRNA half lives and the study of regulated RNA degradation. PUBLIC HEALTH RELEVANCE: Current methodology to study the degradation rates of RNAs in a cell contains procedural bias and is not reliable. While homebrew methods are available they often waste lab personnel time performing assay optimization and validation. Given the fact that changes in mRNA decay rates are likely responsible for almost half of the regulation of gene expression in a cell the lack of a reliable technology to accurately assess mRNA degradation rates is hampering many efforts. We thus propose a novel method to address these issues.