Enabling RNA Analyses from Formaldehyde-fixed Tissues

启用甲醛固定组织的 RNA 分析

• 批准号: 6935698
• 负责人: RICHARD C CONRAD
• 金额: $ 37.71万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6935698
• 关键词: RNA; biotechnology; crosslink; formaldehyde; gene expression profiling; high performance liquid chromatography; histochemistry /cytochemistry; histology; human tissue; laboratory mouse; microarray technology; nucleic acid amplification techniques; nucleic acid purification; nucleic acid quantitation /detection; polymerase chain reaction; technology /technique development; tissue /cell preparation; tissue /organ preservation; tissue resource /registry

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.

描述（由申请人提供）：该提案的长期目标是允许从固定和包埋的组织中分离RNA用于基因表达分析。组织固定和包埋在临床和研究环境中的广泛使用导致医学院、医院和大学积累了大量固定和包埋组织。将特定或整体基因表达的变化与这些样本相关的形态学、诊断和疾病进展信息关联起来的能力将提供大量信息。挖掘这些数据将极大地帮助我们了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，最终改进诊断和治疗。不幸的是，由于固定和嵌入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。在该提案的第一阶段，我们证明了我们可以从固定和包埋的组织中提取适合 RT-PCR 分析的 RNA，并已开始优化所涉及的程序。我们还建立了对 RNA 修饰的灵敏测定法，并证明固定和嵌入引起的 RNA 修饰可以逆转，至少可以部分逆转。在第二阶段，我们将开发产品和程序，以便对固定和包埋的组织样本进行基因表达分析。我们的具体目标是： 1. 继续开发程序以提高固定样本（尤其是档案样本）中功能性 RNA 的质量和数量。 2. 通过修改逆转录反应的反应条件和酶本身，开发一个强大的程序来扩增从甲醛固定的组织中获得的 RNA。 3. 确定从这些样本中获取 RNA 的最佳固定和嵌入参数，并将此信息提供给医学界。 基于这些目标，我们将开发和发布用于 RNA 提取、逆转修饰、逆转录和 RNA 扩增的试剂盒、试剂和改进方案，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。