Fixation of breast and prostate cancer tissue

乳腺癌和前列腺癌组织的固定

• 批准号: 6439088
• 负责人: CHRISTOPHER B UMBRICHT
• 金额: $ 16.35万
• 依托单位: JOHNS HOPKINS 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6439088
• 关键词: DNA; RNA; breast neoplasms; crosslink; gene expression; human tissue; immunocytochemistry; method development; neoplasm /cancer genetics; polynucleotides; prostate neoplasms; tissue /cell preparation

DESCRIPTION (provided by applicant): Comprehensive analysis of gene expression is rapidly becoming a possibility not only for selected model systems, but also for clinical tissue samples. The analysis of gene expression in clinical tissue samples has been severely limited, however, by the standard practice of formalin-based fixation in surgical pathology. Formalin impacts the quality and quantity of cellular macromolecules that can be extracted from fixed tissue. Therefore, any quantitative analysis has historically depended on excess tissue, which could be obtained without compromising the pathologic evaluation. As a result, there is almost no material available from small samples, thus effectively precluding analysis of precancerous lesions and early invasive cancer, both of which may hold the key to many basic questions about the early carcinogenic process. Paradoxically, we are experiencing an unprecedented increase in the accrual of these small lesions thanks to increasing successes in screening programs for both breast and prostate cancer. Clearly, making these specimens available to molecular methods presents a tremendous opportunity to advance our knowledge of the initial phases of carcinogenesis. We propose to address this issue with two complementary approaches. First, improve the currently insufficient methods of RNA and DNA extraction from archival formalin fixed material. Nucleic acid losses due to non-specific trapping in irreversibly crosslinked macromolecules will be minimized by optimizing the breakdown of all non-target components as well as using the affinity of nucleic acids for silica beads in high salt solutions. The template function of extracted nucleic acids will be improved by removing labile N-methylol adducts from the purified polynucleotides. Second, develop new fixation procedures improving the extractability of amplifyable DNA and RNA templates, while avoiding the difficulties of having to divide limited tissue samples according to various clinical and research needs. We have just completed a pilot project demonstrating that alternative alcohol based fixation produces excellent histopathological and immunohistochemical results without compromising extraction of functional messenger RNA, as assessed by quantitative real-time RT-PCR. Fixation without crosslinking may fulfill both clinical and scientific needs.

描述（由申请人提供）： 基因表达的综合分析正迅速成为可能 不仅适用于选定的模型系统，而且适用于临床组织样品。的 临床组织样品中的基因表达分析已经严重 然而，受限于基于福尔马林的固定的标准实践， 外科病理学 福尔马林影响细胞的质量和数量 可以从固定的组织中提取的大分子。 因此，任何定量分析在历史上都依赖于过剩 组织，这可以获得不损害病理 评价因此，几乎没有材料可从小 样本，从而有效地排除了癌前病变的分析， 早期浸润性癌症，这两者都可能是许多基本问题的关键 关于早期致癌过程。巧合的是，我们正在经历一场 这些小病变的发生率空前增加， 乳腺和前列腺筛查项目越来越成功 癌很明显，使这些标本可用于分子方法 提供了一个巨大的机会，以促进我们的知识， 癌发生的阶段。我们建议从两个方面解决这个问题 互补的方法。第一，改进目前不足的方法， 从档案福尔马林固定材料中提取RNA和DNA。核酸 由于不可逆交联大分子中非特异性捕获而造成的损失 将通过优化所有非目标组件的细分来最大限度地减少， 以及利用核酸对高盐中的二氧化硅珠的亲和力 解决方案提取的核酸的模板功能将得到提高 从纯化的多核苷酸中除去不稳定的N-羟甲基加合物。 第二，开发新的固定程序，提高 可扩增的DNA和RNA模板，同时避免了 根据不同的临床和研究， 需求我们刚刚完成了一个试点项目，展示了这种替代方案 基于酒精的固定产生良好的组织病理学， 免疫组化结果，而不损害功能提取 信使RNA，如通过定量实时RT-PCR评估的。固定，无 交联可以满足临床和科学需要。