Enabling RNA Analyses from Formaldehyde-fixed Tissues

启用甲醛固定组织的 RNA 分析

• 批准号: 6787604
• 负责人: RICHARD C CONRAD
• 金额: $ 24.21万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-15 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6787604
• 关键词: RNA; RNase protection assay; adenosine diphosphate; chemical structure function; chemical synthesis; crosslink; data quality /integrity; formaldehyde; gene expression; genetic transcription; high performance liquid chromatography; laboratory mouse; method development; northern blottings; nucleic acid chemical synthesis; nucleic acid purification; nucleic acid quantitation /detection; polymerase chain reaction; tissue /organ preservation

DESCRIPTION (provided by applicant): The overall goal is to develop a robust procedure for extracting RNA from tissues that have been fixed in formaldehyde, either neutral buffered formalin or buffered paraformaldehyde. Clinically interesting tissue from biopsies and necropsies is routinely archived after formaldehyde fixation by medical institutions. The primary use for these tissues is histological, as the RNA and other macromolecular components are so heavily crosslinked that they cannot be retrieved intact. The ability to use these samples in mRNA expression studies presents a huge opportunity. Procedures currently employed are inefficient and often are difficult to reproduce, so improvements are desired. A procedure is sought that provides RNA that is fully functional as a template for reverse transcription and can function in more classical analyses like Northern blots and ribonuclease protection assays (RPA's). Enhancement of the current extraction procedures by utilizing a mixture of hydrolytic enzymes and denaturants, to obtain RNA that has a normal appearance on electrophoresis will be pursued, as well as investigating methodologies for reversing the formaldehyde reaction. In the latter endeavor, methylene-N6-bis-adenosine, a common formaldehyde-generated crosslink, will be synthesized to be used as a test compound under controlled chemical tests to determine conditions for specifically removing the crosslink. This procedure can then be applied to RNA, either in situ (posthomogenization) or after extraction from tissue. Downstream procedures to analyze the RNA obtained will be extensively investigated, concentrating on reverse transcription (cDNA)-based methods. Much of this work will involve comparing the relative levels of different mRNA species inferred by these methods, especially between tissues split before fixing or freezing, to ensure the procedure does not bias the populational representation.

描述（由申请人提供）：总体目标是开发一种稳健的程序，用于从固定在甲醛（中性缓冲福尔马林或多聚甲醛）中的组织中提取RNA。来自活检和尸检的临床上感兴趣的组织通常由医疗机构在甲醛固定后存档。这些组织的主要用途是组织学，因为 RNA 和其他大分子成分高度交联，无法完整回收。在 mRNA 表达研究中使用这些样本的能力提供了巨大的机会。目前采用的程序效率低下并且通常难以重现，因此需要改进。寻求一种程序，提供功能齐全的RNA作为逆转录模板，并且可以在更经典的分析中发挥作用，例如Northern印迹和核糖核酸酶保护测定（RPA）。将寻求通过利用水解酶和变性剂的混合物来增强当前的提取程序，以获得在电泳上具有正常外观的RNA，并研究逆转甲醛反应的方法。在后一项努力中，将合成亚甲基-N6-双-腺苷（一种常见的甲醛产生的交联），用作受控化学测试中的测试化合物，以确定专门去除交联的条件。然后可以将该程序应用于原位（匀浆后）或从组织中提取后的 RNA。将广泛研究分析所获得的 RNA 的下游程序，重点是基于逆转录 (cDNA) 的方法。这项工作的大部分内容将涉及比较通过这些方法推断的不同 mRNA 种类的相对水平，特别是在固定或冷冻之前分裂的组织之间，以确保该程序不会使群体代表性产生偏差。