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Identification of Novel Prostate Cancer-Causitive Gene Mutations by Representational Difference Analysis of Microdissected Prostate Cancer

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Annual rept. 15 Aug 1999-14 Feb 2001

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The representational difference analysis RDA technique, and its variant the differential subtraction chain DSC technique were attempted on 10 paired samples of microdissected benign and malignant prostate epithelium. The resulting subtraction products were cloned and screened in Southern blots of the initial tester and driver amplicon populations. Truly subtracted products were sequenced and tested for markers of homozygous deletion in the original tissue samples by polymerase chain reaction. No homozygous deletions were found. The research strategy was changed to take advantage of the newly available technology of oligonucleotide microarrays, which have the potential to also achieve the aims of the proposed research. Results of validation studies are given that show that the bulk tissue collected at our institution can identify distinct gene expression clusters between samples of benign and malignant human prostate cancer. Data is given to show that microdissected tissue samples retain RNA of sufficient quality to perform gene expression profiling, and that RNA of the quantity present in microdissected samples can be amplified by an in vitro transcription protocol modified in our laboratory to levels sufficient for microarray analysis.

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  • Genetic Engineering and Molecular Biology
  • Medicine and Medical Research

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