New Methods for Tissue Fixation

组织固定的新方法

• 批准号: 6622110
• 负责人: Louis J Romano
• 金额: $ 14.69万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622110
• 关键词: DNA; Escherichia coli; RNA; bacterial genetics; crosslink; formaldehyde; histochemistry /cytochemistry; messenger RNA; method development; plasmids; polymerase chain reaction; proteins; simian virus 40; tissue /cell culture; tissue /cell preparation; tissues; virus DNA; virus genetics

The long-range goal of this project is to develop new fixation methods for cell preservation that are compatible with molecular analyses used to study cellular proteins, DNA, and RNA. The present application seeks to characterize the structures that are formed between cellular components following treatment with aqueous buffered formaldehyde (neutral buffered formalin), the reagent that is currently the most common fixative agent. It is our contention that a full characterization of these structures will aid in the design of new fixatives and may allow us to develop methods to reverse the formalin linkages. To accomplish this goal we will first use purified systems that model intracellular DNA and RNA to determine the number and types of linkages formed by neutral buffered formalin treatment between proteins and nucleic acids. Once these analytical methods are worked out in these simple systems, they will be extended for formalin-fixed cells. The formalin-fixed model systems will be also be used to provide templates for PCR and RT-PCR analyses to allow a study of how this treatment inhibits these processes. As a part of these studies, attempts will be made to develop methods that might be used to reverse the formaldehyde adducts that have formed to the nucleic acids to generate better templates from fixed cells and a study to determine if the formaldehyde adducts induce instability in RNA. Once the chemistry of formalin fixation and the mechanism of PCR and RT-PCR inhibition are understood, future studies, not part of this application, will use this information to develop new fixative agents that do not interfere with molecular analyses or which can be chemically treated to reverse or cleave the fixation linkages.

该项目的长期目标是开发新的细胞保存固定方法，该方法与用于研究细胞蛋白质、DNA 和 RNA 的分子分析兼容。本申请试图表征用水性缓冲甲醛（中性缓冲福尔马林）处理后细胞成分之间形成的结构，该试剂是目前最常见的固定剂。我们认为，对这些结构的全面表征将有助于设计新的固定剂，并可能使我们能够开发出逆转福尔马林连接的方法。为了实现这一目标，我们将首先使用模拟细胞内 DNA 和 RNA 的纯化系统来确定蛋白质和核酸之间通过中性缓冲福尔马林处理形成的连接的数量和类型。一旦这些分析方法在这些简单的系统中得到解决，它们将扩展到福尔马林固定细胞。福尔马林固定模型系统还将用于提供 PCR 和 RT-PCR 分析模板，以便研究这种处理如何抑制这些过程。作为这些研究的一部分，我们将尝试开发可用于逆转已与核酸形成的甲醛加合物的方法，以便从固定细胞中产生更好的模板，并进行研究以确定甲醛加合物是否会导致 RNA 不稳定。一旦了解了福尔马林固定的化学原理以及 PCR 和 RT-PCR 抑制的机制，未来的研究（不属于本申请的一部分）将使用这些信息来开发新的固定剂，这些固定剂不会干扰分子分析或可以进行化学处理反转或断开固定连杆。