Retrieval of DNA, RNA, and Protein from Archival Tissue

描述(由申请人提供)： 目前的第一阶段R21研究已经实现了三个具体目标。证明了用改进的抗原提取(AR)方法从福尔马林石蜡标本中提取蛋白质、RNA和DNA的可行性。成功地采用了基于PCR的方法来扩增和定量从福尔马林石蜡组织中提取的RNA/DNA。开发了三个模型系统来监测改进后的AR过程的效果，包括新的模拟或人造组织和纯化的“蛋白质基质颗粒”。在这份R33提案中继续这些研究的重点是将这些进展纳入到对作为福尔马林-石蜡块保存的癌症组织进行分子分析的“综合系统方法”中。目前，基于免疫组织化学(IHC)、原位杂交(ISH)和聚合酶链式反应(PCR)的检测方法被广泛应用于福尔马林-石蜡癌组织的常规诊断和基础癌症研究，但由于缺乏统一的方法，特别是完全缺乏标准参考物质，结果的重复性和有效性受到严重质疑，这被认为是在诊断临床实验室使用类似方法所必需的。本提案涉及应用于福尔马林-石蜡癌组织的基于IHC、ISH和PCR的检测的分析前、分析和分析后方面。这一方法的一个组成部分是为被用作或可能被用作癌症或预后标志物的关键分析物制定参考标准，根据这些标准对福尔马林-石蜡癌组织的分析结果进行校准，以产生用于诊断和研究目的的严格定量结果。为了实现组织切片的客观定量目标，将在南加州大学图像核心进行光谱图像分析，为此，我们已经证明了在同一福尔马林-石蜡切片中同时定量分析多达四种不同分析物的可行性。这项提议是多学科的，包括与洛杉矶儿童医院的南加州大学细胞成像核心、帕萨迪纳的亨廷顿医学研究所、罗氏分子系统公司、国家标准与技术研究所(NIST)和加州大学旧金山分校的合作。

DESCRIPTION (provided by applicant): The current phase I R21 study has accomplished three Specific Aims. The feasibility of retrieval/extraction of protein, RNA, and DNA from archival formalin-paraffin tissues by modified Antigen Retrieval (AR) methods was demonstrated. PCR based methods were adapted and applied successfully for the amplification and quantitation of RNA/DNA extracted by modified AR from formalin-paraffin tissues. Three model systems were developed to monitor the efficacy of the modified AR process, including novel simulated or 'faux tissues' and purified 'protein matrix pellets'. Continuation of these studies in this R33 proposal focuses upon the incorporation of these advances into an 'integrated systems approach' to the molecular analysis of cancer tissues that have been preserved as formalin-paraffin blocks. Currently, immunohistochemical (IHC), in situ hybridization (ISH) and PCR based assays are widely applied to formalin-paraffin cancer tissues in 'routine diagnosis' and in basic cancer research, but the reproducibility and the validity of the findings are open to serious question due to the lack of uniform methods, in particular the total lack of 'standard reference materials', which are considered essential to the use of analogous methods in the diagnostic clinical laboratory. This proposal addresses pre-analytical, analytical, and post-analytical aspects of the use of IHC, ISH and PCR based assays as applied to formalin-paraffin cancer tissues. Integral to this approach is the development of reference standards for key analytes that are, or may be, employed as cancer or prognostic markers, against which results obtained by the analysis of formalin-paraffin cancer tissues will be calibrated to yield strict quantitative findings for diagnostic and research purposes. To meet the goal of objective quantitation in tissue sections, spectral image analysis will be performed at the USC Image Core, for which we have shown the feasibility of quantitative analysis of up to four different analytes simultaneously in the same formalin-paraffin section. This proposal is multi-disciplinary, including collaborations with the USC Cellular Imaging Core at Childrens Hospital of Los Angeles, the Huntington Medical Research Institute in Pasadena, Roche Molecular Systems Inc., the National Institute of Standards and Technology (NIST), and the University of California, San Francisco.