The objective of this new Facility Core is to provide Center investigators a wide range of reliable and validated assays to analyze changes in gene expression and to provide informatics support for the storage and analysis of large arrays of data.
A specific aim of this Core is to provide to Center members high throughput assays (examining genome-wide changes), such as cDNA microarrays and serial analysis of gene expression (SAGE), as well as the medium throughput assays (examining tens to hundreds of genes), such as rapid analysis of gene expression (RAGE) and real-time PCR.
Other aims are to cross validate these technologies, to obtain and maintain appropriate cDNA and primer libraries, and to obtain and/or develop bioinformatics support. The grant application stated that this Core plans to provide the following support to Center members: 1) use of external and internal database; 2) training and support for SAGE, RAGE, microarrays, and real-time PCR; 3) validated real-time PCR primers and probes, RAGE primers and linkers, microarray chips (mouse and rat initially, add fish and human if needed/warranted) and fluorescent probes, SAGE clones for sequencing; 4) QC check for cDNA preparations; 5) storage of global expression data; 6) turnkey SAGE, cDNA microarray, RAGE, and real-time PCR experiments, with the members providing the cDNA preparations and the Core the final data. In addition, this Core plans to establish the reliability and validity of the various assays; develop methods for assaying small samples obtained from microdissection; develop and maintain a library of RAGE primers and adapters and a library of real-time PCR assays; and develop computer methods to automate experimental design, data gathering, analysis and presentation of global expression data. This Core will build upon the pre-existing expertise of Drs. MacLeod, Aldaz and DiGiovanni. Dr. MacLeod is the primary inventor of RAGE. The RAGE technology has been used to detect changes in gene expression in murine kereatinocytes overexpressing the E2F1 gene in transgenic mice, compared to the expression in wildtype mice. It has also been used to examine DNA damage response of human mammary epithelial cell HME87 to treatment with BPDE and of mouse skin treated with either BPDE or DMBA. This technology has been transferred to other CRED members and to an external biotech company, with a commercial RAGE software under development. Dr. Aldaz has successfully used the SAGE technology to evaluate changes in gene expression in MCF-7 cells upon treatment with estrogen. Dr. Aldaz has generated SAGE libraries from normal breast tissues, breast tumors, and breast cancer cell lines. Breast cancer and normal breast libraries have been donated to the Cancer Genome Anatomy Project's SAGE database. Dr. DiGiovanni has used Clontech low density cDNA array and Genome Systems Gem-1 higher density cDNA array successfully to characterize transgenic animals.
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