Formalin Fixation and Recovery of RNA and Protein

福尔马林固定以及 RNA 和蛋白质的回收

• 批准号: 6522670
• 负责人: TIMOTHY J. O'LEARY
• 金额: $ 11.24万
• 依托单位: AMERICAN REGISTRY OF PATHOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6522670
• 关键词: circular dichroism; enzyme linked immunosorbent assay; formaldehyde; human tissue; messenger RNA; method development; microcalorimetry; polymerase chain reaction; protein structure; proteins; tissue /cell preparation; ultracentrifugation

To understand the molecular pathogenesis of cancer, one must relate genetic changes, such as mutation or altered expression, to metastasis, treatment response and survival. When the clinical course is typically short (for example, pancreatic cancer), it may be possible to achieve these objectives using fresh or frozen issue, for which molecular biologic and protein analyses are relatively straightforward. Unfortunately, many cancers (such as breast cancer) have a course in which many years may elapse between treatment of the primary tumor and the appearance of metastasis. The use of formalin-fixed paraffin-embedded archival issues for molecular and proteomic analysis could reduce the time to obtain clinical correlations, but is not currently feasible for many types of investigations. For example, quantities of "accessible" RNA in archival tissue are currently insufficient to permit analysis using such techniques as SAGE or nucleic acid microarrays. the objectives of this grant are to develop a deeper understanding of protein and nucleic acid fixation by formalin, using this understanding to develop improved methods by which mRNA and proteins may be made available for proteomic and genomic analysis. We will determine the effect of formalin fixation and standard antigen-retrieval protocols on protein secondary structure using CD spectroscopy and differential scanning microcalorimetry, on protein tertiary structure using analytical ultracentrifugation, and on protein antigenicity using ELISA assays. We will further determine the conditions required for the chemical reversal of protein formalin fixation. Similarly, we will determine the effects of methylol and methylol-amino acid adduct formation with nucleic acid bases on the efficiency of RT-PCR based methods, and determine the conditions under which these adducts may be efficiently and effectively removed from nucleic acids. Achievement of these objectives will effectively advance us towards the broader objective of developing retrieval conditions that both enable extraction of intact proteins from fixed tissue sections for proteomic analysis, and extraction of intact mRNA transcripts for application in serial analysis of gene expression (SAGE) and DNA microarray applications.

要了解癌症的分子发病机制，必须将基因变化（如突变或表达改变）与转移、治疗反应和存活率联系起来。当临床病程通常较短时（例如胰腺癌），可以使用新鲜或冷冻组织实现这些目标，分子生物学和蛋白质分析相对简单。不幸的是，许多癌症（如乳腺癌）的病程中，原发性肿瘤的治疗和转移的出现之间可能会经过多年。使用福尔马林固定石蜡包埋的档案问题的分子和蛋白质组学分析可以减少时间，以获得临床相关性，但目前是不可行的许多类型的调查。例如，档案组织中“可接近的”RNA的量目前不足以允许使用诸如SAGE或核酸微阵列的技术进行分析。这项资助的目的是加深对福尔马林固定蛋白质和核酸的理解，并利用这种理解开发改进的方法，使mRNA和蛋白质可用于蛋白质组学和基因组学分析。我们将使用CD光谱和差示扫描微量热法确定福尔马林固定和标准抗原修复方案对蛋白质二级结构的影响，使用分析超离心法确定蛋白质三级结构的影响，以及使用ELISA测定法确定蛋白质抗原性的影响。我们将进一步确定蛋白质福尔马林固定的化学逆转所需的条件。类似地，我们将确定羟甲基和羟甲基-氨基酸加合物与核酸碱基的形成对基于RT-PCR的方法的效率的影响，并确定这些加合物可以有效地和有效地从核酸中去除的条件。这些目标的实现将有效地推进我们对更广泛的目标，开发检索条件，使完整的蛋白质从固定的组织切片提取蛋白质组学分析，并提取完整的mRNA转录的应用程序中的基因表达的系列分析（SAGE）和DNA微阵列应用。