Gene expression profile during Ebselen treatment in budding yeast

Ebselen is synthetic selenium containing organic compound. It exhibits glutathione peroxidase (GSH-Px) like activity hence acts as a potent antioxidant therefore has been widely used in the treatment of various diseases. In contrast, certain studies also demonstrate its toxic nature; however its mechanism of action is not well understood. Presently, Ebselen has been used at 100 µM concentration for the elucidation of its molecular and cellular mechanism of action. Microarray analysis identified the differentially expressed genes in 100 µM Ebselen concentration. Total RNA was isolated from 100 µM Ebselen and DMSO (control) treated yeast (Saccharomyces cerevisiae) cell culture by heat/freeze method as described previously (Schmitt et al., 1990) with slight modification. Whole genome expression was performed on Affymetrix GeneChipYeast Genome 2.0 Arrays (Affymetrix, Santa Clara, CA) as described in GeneChip® 3’ IVT Express Kit User Manual. Briefly, RNA was reverse transcribed to synthesize First-Strand cDNA that was primed with T7 oligo (dT) primer to synthesize cDNA containing a T7 promoter sequence. Second-strand cDNA Synthesis was performed to convert the single-stranded cDNA into double stranded DNA (dsDNA) which acted as a template for transcription. The reaction employed DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. In vitro transcription was performed to synthesize biotin-modified aRNA with IVT labeling master mix generates multiple copies of biotin-modified aRNA from the double stranded cDNA templates; this is the amplification step. aRNA Purification removes unincorporated NTPs, salts, enzymes, and inorganic phosphate to improve the stability of the biotin-modified aRNA. Fragmentation of labeled aRNA prepares the target for hybridization to GeneChip® 3’ expression arrays. The labeled cRNA was purified, fragmented, and hybridized to the arrays at 45°C for 16 h with constant rotational mixing at 60 rpm. Washing and staining of the arrays was performed using the Affymetrix GeneChip Fluidics Station 450. Arrays were scanned using an Affymetrix GeneChip Scanner 3000 and GCOS software. The data set was analyzed using trial version of Gene Spring software 7.2 (Silicon Genetics, Palo Alto, CA). The Affymetrix CEL files were normalized. Statistically significant differentially expressed genes were identified through t-test with a Benjamini & Hochberg test correction.