Enabling RNA Analyses from Formaldehyde-fixed Tissues

描述（由申请人提供）：总体目标是开发一种强大的程序，用于从已固定在甲醛中的组织中提取RNA，无论是中性缓冲福尔马林还是缓冲多聚甲醛。在医疗机构进行甲醛固定后，临床感兴趣的活检和尸检组织通常会存档。这些组织的主要用途是组织学，因为RNA和其他大分子成分是如此严重的交联，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一个巨大的机会。目前采用的程序效率低下，而且往往难以复制，因此需要改进。我们正在寻找一种方法，提供一种功能齐全的RNA作为反转录模板，并能在更经典的分析中发挥作用，如Northern blots和核糖核酸酶保护试验（RPA）。通过使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。在后一种努力中，将合成亚甲基- n6 -二腺苷，一种常见的甲醛生成的交联，作为受控化学测试的测试化合物，以确定具体去除交联的条件。然后，该程序可以应用于RNA，无论是原位（后同源化）还是从组织中提取后。下游程序分析获得的RNA将被广泛研究，集中在反转录（cDNA）为基础的方法。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物种的相对水平，特别是在固定或冷冻前分裂的组织之间，以确保该过程不会使总体代表性产生偏差。

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.