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Abstract

Grant Number:	5R21CA091227-02
Project Title:	Formalin Fixation and Recovery of RNA and Protein

PI Information:	Name	Email	Title
	O'LEARY, TIMOTHY J.	timothy.oleary@hq.med.va.gov	DIRECTOR

Abstract: To understand the molecular pathogenesis of cancer, one must relate genetic changes, such as mutation or altered expression, to metastasis, treatment response and survival. When the clinical course is typically short (for example, pancreatic cancer), it may be possible to achieve these objectives using fresh or frozen issue, for which molecular biologic and protein analyses are relatively straightforward. Unfortunately, many cancers (such as breast cancer) have a course in which many years may elapse between treatment of the primary tumor and the appearance of metastasis. The use of formalin-fixed paraffin-embedded archival issues for molecular and proteomic analysis could reduce the time to obtain clinical correlations, but is not currently feasible for many types of investigations. For example, quantities of "accessible" RNA in archival tissue are currently insufficient to permit analysis using such techniques as SAGE or nucleic acid microarrays. the objectives of this grant are to develop a deeper understanding of protein and nucleic acid fixation by formalin, using this understanding to develop improved methods by which mRNA and proteins may be made available for proteomic and genomic analysis. We will determine the effect of formalin fixation and standard antigen-retrieval protocols on protein secondary structure using CD spectroscopy and differential scanning microcalorimetry, on protein tertiary structure using analytical ultracentrifugation, and on protein antigenicity using ELISA assays. We will further determine the conditions required for the chemical reversal of protein formalin fixation. Similarly, we will determine the effects of methylol and methylol-amino acid adduct formation with nucleic acid bases on the efficiency of RT-PCR based methods, and determine the conditions under which these adducts may be efficiently and effectively removed from nucleic acids. Achievement of these objectives will effectively advance us towards the broader objective of developing retrieval conditions that both enable extraction of intact proteins from fixed tissue sections for proteomic analysis, and extraction of intact mRNA transcripts for application in serial analysis of gene expression (SAGE) and DNA microarray applications.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
formaldehyde, method development, tissue /cell preparation
messenger RNA, protein, protein structure
circular dichroism, enzyme linked immunosorbent assay, human tissue, microcalorimetry, polymerase chain reaction, ultracentrifugation

Institution:	AMERICAN REGISTRY OF PATHOLOGY, INC.
	WASHINGTON, DC 20306
Fiscal Year:	2002
Department:
Project Start:	01-AUG-2001
Project End:	31-JUL-2004
ICD:	NATIONAL CANCER INSTITUTE
IRG:	ZCA1

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