Enabling RNA Analyses from Formaldehyde-fixed Tissues

描述(由申请人提供)：这项建议的长期目标是允许从固定和嵌入的组织中分离RNA，用于基因表达分析。组织固定和埋入在临床和研究环境中的广泛使用导致医学院、医院和大学中积累了大量固定和埋入的组织。将特定或整体基因表达的变化与与这些样本相关的形态、诊断和疾病进展信息相关联的能力将是非常有用的。挖掘这些数据将极大地帮助我们理解特定基因在疾病和发育中的作用，并将有助于识别各种人类疾病的标记和致病基因，最终导致改进诊断和治疗。不幸的是，由于固定和包埋过程引起的交联和化学修饰，这些样品中的RNA实际上无法用于这些研究。在这项计划的第一阶段，我们证明了我们可以从固定的和包埋的组织中提取适合RT-PCR分析的RNA，并已经开始优化涉及的程序。我们还建立了敏感的RNA修饰检测方法，并证明了固定和包埋引起的RNA修饰至少部分是可以逆转的。在第二阶段，我们将开发产品和程序，允许对固定和嵌入的组织样本进行基因表达分析。我们的具体目标是： 1.继续制定程序，以提高从固定样本，特别是从档案中提取功能RNA的质量和数量。 2.建立一种稳健的方法来扩增从甲醛固定的组织中获得的RNA，通过改变反应条件和酶本身来进行逆转录反应。 3.确定从这些样本中提取RNA的最佳固定和包埋参数，并将此信息提供给医学界。 从这些目标出发，我们将开发和发布用于RNA提取、修饰逆转、反转录和RNA扩增的试剂盒、试剂和改进的方案，以及改进的固定和包埋程序，以更好地从这些组织中回收RNA。

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to permit the isolation of RNA from fixed and imbedded tissues for gene expression analysis. The widespread use of tissue fixation and embedding in both clinical and research settings has resulted in the accumulation of large stores of fixed and embedded tissues in medical schools, hospitals, and universities. The ability to correlate changes in either specific or global gene expression with the morphological, diagnostic and disease progression information associated with these samples would be tremendously informative. Mining this data would greatly aid our understanding of the roles of specific genes in disease and development and would help identify markers and causative genes for a variety of human diseases, ultimately leading to improved diagnostics and therapeutics. Unfortunately, the RNA in these samples has been practically unavailable for these studies, due to the crosslinking and chemical modifications induced by the fixing and embedding processes. During Phase I of this proposal, we demonstrated that we can extract RNA suitable for RT-PCR analysis from fixed and embedded tissues and have begun optimizing the procedures involved. We also established sensitive assays for modifications to the RNA and have demonstrated that the modifications to RNA induced by fixation and embedding can be reversed, at least partially. Under Phase II we will develop products and procedures to allow gene expression analysis from fixed and embedded tissue samples. Our specific aims are: 1. Continue to develop procedures to increase the quality and quantity of functional RNA from fixed samples, especially from archives. 2. Develop a robust procedure to amplify RNA obtained from formaldehyde-fixed tissue, by modifying both the reaction conditions and enzyme itself for the reverse transcription reaction. 3. Determine optimal fixing and embedding parameters for obtaining RNA from these samples and provide this information to the medical community. From these aims, we will develop and release kits, reagents, and improved protocols for RNA extraction, reversal of modifications, reverse transcription and RNA amplification, and improved fixation and embedding procedures to better reclaim RNA from these tissues.