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

描述（由申请人提供）：虽然mRNA衰变率的变化约占细胞中基因表达调控的50%，但目前尚无准确可靠地测量mRNA半衰期的测定方法。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mRNA衰减率，这大大减缓了该领域的进展。为了满足这一需求，我们开发了一种新的方法，包括对代谢标记的RNA分子进行直接PCR分析，以确定mRNA的衰变速率。在这个应用中，我们提出在两个目标上证明这种新方法的可行性并验证其有效性。首先，我们将优化一种独特的核苷类似物代谢标记技术，该技术利用简单且经过历史验证的偶联化学方法，结合选择性RT-PCR扩增所需mRNA群体。其次，我们将使用合成的mrna和几种具有良好特征的内源性细胞mrna验证我们的代谢标记技术/RT-PCR方法。总的来说，该试剂盒将提供一种无偏见、用户友好、可靠的方法，用于常规测定mRNA半衰期和研究受调节的RNA降解。

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.