Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

摘要

The expression pattern of protein tyrosine kinase (tk) genes are often found altered in prostate cancer tissues. We developed a cDNA micro-array- based screening system to measure the expression levels of tk genes. The hardware for preparation of cDNA micro-arrays and basic protocols for hybridization were developed in year 1. In the year 2, we finished cDNA synthesis from prostate cancer cell lines and frozen tissue specimens. We optimized protocols for PCP amplification and cloning and added additional targets to our micro-arrays. Using well-characterized prostate cancer cell lines, the system delivered reproducible results during cell transformation and progression towards a more malignant phenotype. Comparing the absolute expression levels from oDNA microarrays with data from Northern blot analyses suggested that our initial approach using mixed-based oligonucleotide primers led to lowered representation of highly abundant transcripts. Furthermore, the mixed base oligonucleotide primers used in the initial cloning experiments lead to some level of unspecific amplification limiting the assay sensitivity. Problems related to primer specificity were addressed with a new primer design based on the nucleotide sequences of known genes. Furthermore, we developed a 2- step in vitro DNA amplification scheme for unbiased quantitation of tk gene expression levels in small samples.