Identification of Novel Prostate Cancer-Causitive Gene Mutations by Representational Difference Analysis of Microdissected Prostate Cancer
Abstract
Tissue from 23 radical prostatectomy operations has been procured. Microdissection of normal and cancer cells have been done for 2 specimens. Representational difference analysis has been completed on one sample yielding subtraction products which have been cloned and sequenced. Subsequent analysis showed that none of these products were from areas of homozygous deletion from the original tumor specimen. It was suspected that the published RDA protocol was not working properly. A control RDA with "spiked" target DNA sequences confirmed this. Using the same control DNA we have optimized the RDA protocol. The optimized protocol has been applied to two cases of primary prostate cancer, and the subtraction products are currently being analyzed. We have extended one of the stated goals of this project and will attempt to perform RDA on both RNA and DNA samples of primary prostate cancers. In this we can assay for transcriptional as well as genetic changes during prostate tumor progression. Towards this end, we have optimized an RNA extraction procedure from microdissected cells. We show that from as little as 2500 cells obtained from primary human tissue using laser capture microdissection we can isolate high molecular weight RNA suitable for this analysis.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 1999
- Accession Number
- ADA380278
Entities
People
- Christopher Moskaluk
Organizations
- University of Virginia