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SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY
Citation:
Ren, H., K E. Thompson, J E. Schmid, AND D J. Dix. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY. Presented at Society of Toxicology, Salt Lake City, UT, March 09 - 13, 2003.
Description:
Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27711.
DNA microarray technology has been widely used to detect differential gene expression and to generate gene expression profiles useful for studying drug or environmental effects on animals or humans. In order to establish methods for monitoring effects of environmental chemicals on the male reproductive system, we have collected rat (pool of ten 75-90 day Sprague-Dawley rats) epididymal sperm and isolated total RNA. Due to the limited number of sperm from individual animals, the amount of total RNA purified from rat sperm was not sufficient for microarray probe labeling. Therefore, RNA amplification was used to generate sufficient quantities of antisense RNA (aRNA) for array hybridization. Two rounds amplification of 200ng rat sperm RNA generated 25 to 35ug aRNA. Only one round of amplification was necessary to obtain similar amounts of aRNA from 200ng testis RNA. After indirect labeling with Cy3 or Cy5, aRNAs were hybridized to glass-slide microarrays with 70-mer oligonucleotides probes for 4,370 rat genes. Preliminary analysis identified hundreds of genes/mRNAs in both the testis and sperm samples (e.g., SP22 sperm protein). These results indicate that RNA amplification can be used to generate gene expression profiles from tissue samples with limited amounts of RNA. More detailed studies of sperm RNA profiles from rats following chemical exposure are underway. Results from these toxicology studies promise to complement studies of sperm RNA profiles in humans.
This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.