Recent progress in large scale DNA sequencing has brought access to all human genes within view. Elucidating which genes are most important in processes such as cell death, proliferation, and differentiation remains a daunting task that will require new strategies and technologies. Among the most valuable strategies to date has been the identification of those genes that are differentially expressed under various physiological or pathophysiological conditions. Unfortunately, there are no rapid, reliable methods to identify these genes. This study will extend our very preliminary work performing large scale differential mRNA expression analysis and evaluate differential gene expression in human endothelial cells undergoing apoptosis. To date, we have devised a protocol to label a small quantity of renal epithelial cell RNA to very high specific activity and used it to probe a commercially available membrane spotted with an array of 27,648 cDNAs whose partial sequences are available in the expressed sequence tags (EST) database. Using this reverse northern analysis we observed a pattern of tissue-specific mRNA expression. The overall goals are to 1) complete experiments validating the specificity and sensitivity of our method and 2) use this improved reverse northern method to identify differentially expressed genes in HUVECs stimulated to undergo apoptosis. The results of this work will be important to the mission of the Harvard Skin Disease Research Center for several reasons. This project is explicitly focused on making large scale differential gene expression analysis available to all investigators within the skin disease field. Further, it will enable investigators to begin to identify and obtain those select genes of the 75,000 expressed genes that are likely to be key players in important biological processes. Finally, by choosing to study apoptosis in endothelial cells, we will identify genes of potential significance for understanding vasculitis, wound healing, autoimmune diseases, anti-phospholipid disease, and other biologically important facets of skin disease.
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