Fixation of breast and prostate cancer tissue

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，也适用于临床组织样本。这个 临床组织样本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做法 外科病理学。福尔马林对细胞质量和数量的影响 可以从固定组织中提取的大分子。 因此，任何量化分析在历史上都依赖于过度 组织，可以在不损害病理的情况下获得 评估。因此，几乎没有任何小规模的材料可用 样本，从而有效地排除了对癌前病变和 早期浸润性癌症，两者都可能是许多基本问题的关键 关于早期的致癌过程。矛盾的是，我们正在经历一场 这些小病变的累积量空前增加，这要归功于 乳腺癌和前列腺癌的筛查项目越来越成功 癌症。显然，让这些样本可用于分子方法 提供了一个极好的机会来提高我们对初始 癌症发生的阶段。我们建议通过两个方面来解决这个问题 互补的方法。一是改进目前存在的不足之处 从福尔马林固定材料中提取RNA和DNA。核酸 不可逆交联大分子的非特异性捕获损失 将通过优化所有非目标组件的细分来最小化 以及利用核酸在高盐中对硅珠的亲和力 解决办法。提取的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。 第二，开发新的固定程序，提高可萃取性 可扩增的DNA和RNA模板，同时避免了 根据不同的临床和研究划分有限的组织样本 需要。我们刚刚完成了一个试点项目，展示了这一替代方案 基于酒精的固定可产生良好的组织病理学和 免疫组织化学结果在不影响功能提取的情况下 信使RNA，用实时定量RT-PCR检测。不带固定装置 交联剂可以同时满足临床和科学的需要。

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.