Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


WELSH

The RAP-array approach to cDNA array hybridization

John Welsh

Sidney Kimmel Cancer Center, San Diego, California, USA

Transcript abundance in biological samples vary typically from many thousands to fewer than one copy per cell. An important practical concern with current microarray technology is the reliable detection of signals from rare transcripts. To address this problem, optimizations in dyes, detection systems, hybridization conditions, attachment strategies, and other variables are on-going in many laboratories. An alternative solution involves methods for the construction of labeled cDNAs with distorted ratios of individual sequences such that their representation is decoupled from their original abundances. In one such approach RNA arbitrarily primed-PCR fingerprinting or Differential Display is used to create probes with reproducibly altered abundances. Due to the selectivity of arbitrary priming, sequences in the low abundance-high complexity class are more highly represented in the resulting cDNA probe relative to transcripts in the higher abundance classes. Within a single sample, sequence abundances are highly distorted relative to the corresponding abundances in the original RNA population. However, this distortion is reproducible on a sequence-by-sequence basis, so that original ratios of transcript abundances are preserved in comparisons between two or more RNA sources.

In our expression profiling studies, we have encountered several interesting new phenomena. A new EGF and TGF-beta regulated gene, VAV3, and a natural 5'-trunkated variant, VAV3.1, will be discussed. Also, several phenomena have emerged from expression profiling of combinatoric treatment experiments, including a vector/vector complement pattern, a pattern characteristic of translation inhibition, and a particularly puzzling pattern that implies that UVC irradiation overrides the well-documented repressive effect that cycloheximide has on RNA turnover.

 


Return to Table of Contents