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

开发新合成 RNA 分析新方法

• 批准号: 8201509
• 负责人: LANCE P FORD
• 金额: $ 17.77万
• 依托单位: BIOO SCIENTIFIC CORPORATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201509
• 关键词: Accounting; Address; Affect; Area; Asses; Biological Assay; Biomedical Research; Cells; Cellular Stress Response; Chemicals; Chemistry; Communities; Complementary DNA; Coupled; Dactinomycin; Development; Effectiveness; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Gold; Growth; Half-Life; Human; Human Resources; Individual; Label; Mammalian Cell; Marketing; Measures; Messenger RNA; Metabolic; Methodology; Methods; Minority; Molecular Biology; Nucleosides; Oncogenic; Pathway interactions; Play; Population; Quality Control; RNA; RNA Decay; RNA Degradation; RNA chemical synthesis; RNA-Directed DNA Polymerase; Reaction; Regulation; Reporting; Reproducibility; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Stimulus; Techniques; Technology; Tetanus Helper Peptide; Thiouridine; Time; Transcript; Uridine; Validation; base; cell killing; design; gene cloning; inhibitor/antagonist; interest; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; meetings; novel; novel strategies; nucleoside analog; prevent; response; user-friendly; wasting

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.

描述（由申请人提供）：虽然mRNA衰减率的变化约占细胞中基因表达调控的50％，但目前尚不可用地准确且可靠地测量mRNA Half Lives的测定法。特别是，可靠的商业试剂盒的缺乏使平均生物医学研究实验室能够有效研究mRNA衰变率正在显着降低该领域的进展。为了满足这种需求，我们开发了一种新型方法，涉及代谢标记的RNA分子的直接PCR分析以确定mRNA衰减率。在此应用中，我们建议证明这种新颖方法的可行性，并在两个目标中验证其有效性。首先，我们将优化一种独特的核苷模拟代谢标记技术，该标记技术利用一种简单且经过历史验证的共轭化学方法以及选择性的RT-PCR扩增所需的mRNA种群。其次，我们将使用合成mRNA和几种特征良好的内源性细胞mRNA验证我们的代谢标记技术/RT-PCR方法。该套件共同使无偏见的，用户友好，可靠的方法常规确定mRNA Half Lives和调节RNA降解的研究。 公共卫生相关性：研究细胞中RNA降解率的当前方法包含程序偏见，并且不可靠。虽然可以使用自制方法，但它们通常会浪费实验室人员的时间进行测定优化和验证。鉴于mRNA衰减率的变化可能导致细胞中基因表达调节的几乎一半，因此缺乏可靠的技术来准确评估mRNA降解速率正在妨碍许多努力。因此，我们提出了一种解决这些问题的新方法。