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衰变率的变化约占细胞基因表达调节的50%，但目前还没有准确可靠地测量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衰变率，这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，涉及直接PCR分析代谢标记的RNA分子，以确定mRNA的衰减率。在这个应用程序中，我们建议证明这种新方法的可行性，并验证其有效性在两个目标。首先，我们将优化一种独特的核苷类似物代谢标记技术，该技术利用简单且历史验证的缀合化学方法，并结合所需mRNA群体的选择性RT-PCR扩增。其次，我们将使用合成mRNA和几种表征良好的内源性细胞mRNA来验证我们的代谢标记技术/RT-PCR方法。总的来说，该试剂盒将为mRNA半衰期的常规测定和受调控RNA降解的研究提供无偏倚、用户友好、可靠的方法。 公共卫生相关性：目前研究细胞中RNA降解率的方法存在程序偏倚，不可靠。虽然自制方法是可用的，但它们通常浪费实验室人员进行测定优化和验证的时间。鉴于mRNA降解速率的变化可能对细胞中几乎一半的基因表达调控负责，缺乏准确评估mRNA降解速率的可靠技术阻碍了许多努力。因此，我们提出了一种新的方法来解决这些问题。