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.
