Profiling of Rgt1-regulated gene transcripts (30º C) in the fungal pathogen Candida albicans

C. albicans glucose sensing and signaling is important for full virulence in the mammalian host. To better understand this phenomenon, we investigated the Hgt4 glucose-sensing pathway, focusing on the downstream transcriptional repressor CaRgt1. CaRgt1-regulated gene transcripts were identified by comparing total RNA extracted from wild type and rgt1 mutant strains. Cells were grown at 30 degrees C in the presence of glycerol, conditions under which CaRgt1 was expected to repress gene transcription. Keywords: cell type comparison A C. albicans genespecific microarray consisting of 70-mer oligonucleotides representing 6,346 of the 6,354 predicted ORFs in the annotated C. albicans genome assembly 19 was used. Each ORF was represented by a specific oligonucleotide, and one genome equivalent of this oligo was spotted three times per slide. To identify Rgt1-regulated transcripts, C. albicans cells from the wild type strain BWP17 or the rgt1 mutant strain CM18 were cultured in synthetic medium containing 5% glycerol at 30º C. Cells were harvested and RNA was extracted, labeled, and hybridized to the arrays. Each experiment consisted of a pair-wise competetive hybridization of total RNA samples derived from the wild type and mutant strains and included a reciprocal dye-flip replicate. Since two biological duplicates were tested for each strain, a total of four DNA microarray slides were used. Slides were scanned immediately after hybridization on a ScanArray express HT scanner (Perkin-Elmer). The scanner photomultiplier tube (PMT) values were set for optimal intensity with minimal background. An additional scan was done for each slide (low PMT) with the PMT such that <1% of the elements were saturated in order to characterise spots which were saturated at the higher PMT setting.