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

摘要

Representational difference analysis (RDA) was attempted on microdissected samples of human prostate cancer to identify areas of genomic homozygous deletion. Difference products were not reliably obtained with this procedure starting from DNA obtained from 5000 cells. When difference products were obtained, subsequent analysis showed that none corresponded to areas of homozygous deletion. To pursue an alternative strategy, oligonucleotide microarray analysis to identify genes that are down-regulated in malignant prostate tissue, procedures were evaluated that would allow the implementation of microarray analysis to microdissected prostate tissue samples. It was determined that approximately 10,000 cells is a reasonable compromise between the need to maximize input material and the time required to perform microdissection. Several amplification protocols and procedures were evaluated for efficacy of amplification and quality of oligonucleotide hybridization results. A working protocol is presented that will amplify mRNA from 10,000 cells to 70 ug after 2 rounds of amplification.