Fixation of breast and prostate cancer tissue

乳腺癌和前列腺癌组织的固定

• 批准号: 6622118
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622118
• 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在迅速成为可能 不仅适用于选定的模型系统，还适用于临床组织样本。这 临床组织样本中基因表达的分析受到严重影响 然而，受限于福尔马林固定的标准做法 外科病理学。 福尔马林影响细胞的质量和数量 可以从固定组织中提取的大分子。 因此，任何定量分析历来都依赖于过量的 组织，可以在不损害病理学的情况下获得 评估。结果，小公司几乎没有可用的材料。 样本，从而有效地排除了癌前病变的分析和 早期浸润性癌症，这两者可能是许多基本问题的关键 关于早期致癌过程。矛盾的是，我们正在经历一个 由于这些小病变的累积史无前例地增加 乳腺癌和前列腺筛查计划的成功率不断提高 癌症。显然，使这些样本可用于分子方法 提供了一个巨大的机会来增进我们对最初的知识的了解 癌发生的阶段。我们建议通过两种方式解决这个问题 互补的方法。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的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分解来最小化 以及在高盐条件下利用核酸对硅珠的亲和力 解决方案。提取核酸的模板功能将得到改善 通过从纯化的多核苷酸中去除不稳定的N-羟甲基加合物。 其次，开发新的固定程序，提高可提取性 可扩增的 DNA 和 RNA 模板，同时避免了 根据临床和研究的不同，划分有限的组织样本 需要。我们刚刚完成了一个试点项目，展示了替代方案 基于酒精的固定产生良好的组织病理学和 免疫组化结果而不影响功能提取 信使 RNA，通过定量实时 RT-PCR 进行评估。固定无 交联可以满足临床和科学的需要。