Recovery of RNA from Formalin-Fixed Tissues

高通量的分子生物学和蛋白质组学方法提供了几种有希望的方法来将基因变化，如突变或基因表达改变，与转移、治疗结果和生存联系起来。在最初诊断和治疗之间的间隔时间很长且出现转移的癌症中，如果能够使用福尔马林固定的石蜡包埋(FFPE)组织来代替新鲜或冷冻的标本，将更容易获得临床相关性。大规模的多重技术，如基因表达序列分析(SAGE)和基因芯片方法，对FFPE组织和非固定组织产生的实验结果略有不同。高通量分子生物学和蛋白质组学方法的长期目标提供了几种有希望的方法来将基因变化，如突变或基因表达改变，与转移、治疗结果和生存联系起来。在最初诊断和治疗之间的间隔时间很长且出现转移的癌症中，如果能够使用福尔马林固定的石蜡包埋(FFPE)组织来代替新鲜或冷冻的标本，将更容易获得临床相关性。大规模的多重技术，如基因表达序列分析(SAGE)和基因芯片方法，对FFPE组织和非固定组织产生的实验结果略有不同。我们研究计划的长期目标是使用高通量的分子生物学筛选方法来识别癌症起源和行为的分子和遗传基础。这项建议的目的是确定在组织学组织处理过程中发生的甲醛诱导的核酸化学修饰，并开发逆转这些修饰的方法。我们的中心假设是，在组织加工过程中形成的甲醛加合物和交联键可以通过一系列加热和透析步骤在适当的溶剂化条件下依次逆转。我们根据初步数据提出了这一假设，这些数据表明，在水溶液中，甲醛引起的蛋白质和核酸化学变化的逆转相对容易，但在有机溶剂存在的脱水后就不那么容易了。这些研究的基本原理是，它们的成功完成将为将高通量筛查方法应用于FFPE组织奠定基础。这将导致改进癌症诊断、评估、治疗和预防的实际干预措施，并促进治疗剂的开发。我们的研究具有创新性，因为我们开创了一种新的模型系统(组织替代物)，非常适合于识别在组织加工过程中发生的甲醛诱导的蛋白质和核酸修饰。在这个项目完成时，我们期望对组织组织学过程中发生的甲醛诱导的对mRNA的化学修饰以及最佳逆转这些修饰的方法有一个全面的了解。这些知识将使我们有能力对FFPE组织进行基因组分析，显著扩大我们进行基因组研究的能力，并为实际研究开辟重要的新领域。我们的研究计划是使用高通量的分子生物学筛选方法来确定癌症起源和行为的分子和遗传基础。这项建议的目的是确定在组织学组织处理过程中发生的甲醛诱导的核酸化学修饰，并开发逆转这些修饰的方法。我们的中心假设是，在组织加工过程中形成的甲醛加合物和交联键可以通过一系列加热和透析步骤在适当的溶剂化条件下依次逆转。我们根据初步数据提出了这一假设，这些数据表明，在水溶液中，甲醛引起的蛋白质和核酸化学变化的逆转相对容易，但在有机溶剂存在的脱水后就不那么容易了。这些研究的基本原理是，它们的成功完成将为将高通量筛查方法应用于FFPE组织奠定基础。这将导致改进癌症诊断、评估、治疗和预防的实际干预措施，并促进治疗剂的开发。我们的研究具有创新性，因为我们开创了一种新的模型系统(组织替代物)，非常适合于识别在组织加工过程中发生的甲醛诱导的蛋白质和核酸修饰。在这个项目完成时，我们期望对组织组织学过程中发生的甲醛诱导的对mRNA的化学修饰以及最佳逆转这些修饰的方法有一个全面的了解。这些知识将导致对FFPE组织进行基因组分析的能力，显著扩展我们进行基因组研究的能力，并为实际调查开辟重要的新领域。

High-throughput molecular biologic and proteomic methods provide several promising approaches for relating genetic changes, such as mutation or altered gene expression, to metastasis, to treatmentoutcomes, and to survival. In cancers where the interval between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations would be more readily obtained if formalin-fixed paraffin-embedded (FFPE) tissues could be used instead of fresh or frozen specimens. Large-scale multiplex techniques, such as serial analysis of gene expression (SAGE), and gene chip methods yield experimental results that are somewhat different for FFPE tissue and unfixed tissue. The long-term goal of High-throughput molecular biologic and proteomic methods provide several promising approaches for relating genetic changes, such as mutation or altered gene expression, to metastasis, to treatment outcomes, and to survival. In cancers where the interval between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations would be more readily obtained if formalin-fixed paraffin-embedded (FFPE) tissues could be used instead of fresh or frozen specimens. Large-scale multiplex techniques, such as serial analysis of gene expression (SAGE), and gene chip methods yield experimental results that are somewhat different for FFPE tissue and unfixed tissue. The long-term goal of our research program is to use high-throughput molecular biologic screening methods to identify the molecular and genetic basis of cancer origins and behavior. The objective of this proposal is to identify the formaldehyde-induced chemical modifications that occur to nucleic acids during histologic tissue processing and to develop methods to reverse these modifications. Our centralhypothesis is that formaldehyde adducts and cross-links formed during tissue processing can be sequentially reversed by a series of heating and dialysis steps, carried out under appropriate solvation conditions. We formulated this hypothesis on the basis of preliminary data which show that the reversal of formaldehyde-induced chemical changes in proteins and nucleic acids is relatively facile in aqueous solutions, but less so following dehydration in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for applying high-throughput screening methods to FFPE tissues. This will lead to improved practical interventions for the diagnosis, evaluation, treatment, and prevention of cancer and facilitate the development of therapeutic agents. Our studies are innovative in that we have pioneered a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins and nucleic acids that occur during tissue processing. At the completion of this project it is our expectation to have established a comprehensive understanding of the formaldehyde-induced chemical modifications to mRNA that occur during tissue histology, and methods for optimally reversing these modifications. This knowledge should result in an ability to carry out genomic analysis on FFPE tissue, significantly expanding our capability to conduct genomic research and opening important new areas to practical investigation. our research program is to use high-throughput molecular biologic screening methods to identify the molecular and genetic basis of cancer origins and behavior. The objective of this proposal is to identify the formaldehyde-induced chemical modifications that occur to nucleic acids during histologic tissue processing and to develop methods to reverse these modifications. Our centralhypothesis is that formaldehyde adducts and cross-links formed during tissue processing can be sequentially reversed by a series of heating and dialysis steps, carried out under appropriate solvation conditions. We formulated this hypothesis on the basis of preliminary data which show that the reversal of formaldehyde-induced chemical changes in proteins and nucleic acids is relatively facile in aqueous solutions, but less so following dehydration in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for applying high-throughput screening methods to FFPE tissues. This will lead to improved practical interventions for the diagnosis, evaluation, treatment, and prevention of cancer and facilitate the development of therapeutic agents. Our studies are innovative in that we have pioneered a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins and nucleic acids that occur during tissue processing. At the completion of this project it is our expectation to have established a comprehensive understanding of the formaldehyde-induced chemical modifications to mRNA that occur during tissue histology, and methods for optimally reversing these modifications. This knowledge should result in an ability to carry out genomic analysis on FFPE tissue, significantly expandingour capability to conduct genomic research and opening important new areas to practical investigation.