Enabling RNA Analyses from Formaldehyde-fixed Tissues

描述（由申请方提供）：总体目标是开发一种稳健的方法，用于从甲醛（中性缓冲福尔马林或缓冲多聚甲醛）中固定的组织中提取RNA。来自活组织检查和尸检的临床上感兴趣的组织通常在医疗机构进行甲醛固定后存档。这些组织的主要用途是组织学，因为RNA和其他大分子组分是如此高度交联，以至于它们不能完整地回收。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，往往难以复制，因此需要改进。寻求一种提供RNA的方法，所述RNA作为逆转录的模板是完全功能性的，并且可以在更经典的分析如北方印迹和核糖核酸酶保护测定（RPA）中起作用。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，以及研究逆转甲醛反应的方法。在后一项奋进中，将合成亚甲基-N6-双腺苷（一种常见的甲酰化生成的交联剂），以在受控化学测试下用作测试化合物，以确定特异性去除交联剂的条件。然后可以将该程序应用于RNA，无论是在原位（同源化后）还是从组织中提取后。将广泛研究分析获得的RNA的下游程序，集中于基于逆转录（cDNA）的方法。这项工作的大部分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。

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.