Expression Profiling of Tyrosine Kinase Genes
Annual rept. 1 Aug 2001-31 Jul 2002
CALIFORNIA UNIV BERKELEY LAWRENCE BERKELEY LAB
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The expression of genes involved in signal transduction e.g. protein kinases is often altered in tumors. The aberrant expression of several of these genes typically parallels the progression toward a more malignant phenotype. We developed a cDNA micro-array - based screening system to measure the level of expression of tyrosine kinase tk genes. The hardware for preparation of cDNA micro-arrays and basic protocols for hybridization were developed in year 1. In the second year, we finished isolating RNA and cDNA synthesis from 16 breast cancer cell lines and 10 frozen tissues. We optimized protocols for tk-specific PCR amplification and cloning. We continued our DNA seguencing effort and added additional targets to our micro-arrays. Using well-characterized breast cancer cell lines, the system delivered reproducible results about tk gene expression during cell transformation and progression toward a more malignant phenotype. Comparing the absolute expression levels from cDNA micro-arrays with data from Northern blot analyses suggested that our initial approach using mixed-based oligonucleotide primers led to lowered representation of highly abundant transcripts. This problem has been addressed with a new oligonucleotide primer design and a modified DNA amplification protocol that we now apply to investigate the tk gene expression in small tissue samples.
- Medicine and Medical Research