Formalin Fixation and Recovery of RNA and Protein

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、治疗反应和生存联系起来。当临床病程通常很短时(例如胰腺癌)，使用新鲜或冷冻的问题可能实现这些目标，对于这些问题，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)的病程从原发肿瘤的治疗到出现转移可能需要很多年的时间。使用福尔马林固定的石蜡包埋档案问题进行分子和蛋白质组学分析可以减少获得临床相关性的时间，但目前不适用于许多类型的研究。例如，档案组织中“可获得”的RNA数量目前不足以使用SAGE或核酸微阵列等技术进行分析。这笔赠款的目的是加深对福尔马林固定蛋白质和核酸的了解，利用这种理解开发改进的方法，使mRNA和蛋白质可以用于蛋白质组和基因组分析。我们将使用CD光谱和差示扫描微量热法来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，使用分析性超速离心法来确定蛋白质三级结构的影响，并使用ELISA法来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。同样，我们将确定羟甲基和羟甲基-氨基酸与核酸碱基形成加合物对基于RT-PCR方法的效率的影响，并确定这些加合物可以有效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动我们朝着更广泛的目标发展，即开发检索条件，既能够从固定的组织切片中提取完整的蛋白质用于蛋白质组分析，又能够提取完整的mRNA转录本，用于基因表达序列分析(SAGE)和DNA微阵列应用。

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.