Microarray and Real–time Transcriptional Analyses of Molecular Responses to Oxidative Stress in the Human Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: Microarray and Real–time Transcriptional Analyses of Molecular Responses to Oxidative Stress in the Human Retinal Pigment Epithelium Methods: We analyzed quantitative changes in gene expression in ARPE19 cells following oxidative stress (OS) in vitro using microarray and real–time PCR methods. Confluent cultures of ARPE19 cells were treated with 500µM H₂O₂ (37ºC, 1 hour) and RNA was isolated from replicate cultures (TRIzol) immediately following, and 4 hours after OS. Pooled cellular RNA was cleaned by column chromatography and reverse transcribed using standard methods. cRNA was synthesized, labeled, and hybridized to the Affymetrix U133A GeneChip array, and scanned using a laser confocal scanner. The scanned images were analyzed using the Affymetrix Microarray Suite (V5.0, Genome Explorations, Memphis). DNAse–treated cellular RNA from the same isolate was reverse transcribed and real–time PCR was performed by standard SYBR–Green amplification methods using selected gene–specific primers. Results: Our microarray studies identified OS–induced changes in the expression of more than 2000 genes at a 2–fold, or greater level in ARPE19 cells. As expected, these changes were seen across a broad range of functional classes including genes involved in apoptosis, redox state, repair, signal transduction, transcription factors, chaperones and cytokines. A broad panel of genes that showed significant changes in the level of expression at both time points was selected from each functional class for further analysis by real–time RT–PCR. These studies are in progress and will be reported at the meeting, together with the microarray data. Conclusions: The human RPE demonstrates quantifiable changes in the expression of many genes in response to sublethal oxidative stress in vitro. These stress–specific responses occur across a broad range of functional classes in the cell and may correlate with the severity of the stress.

Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium • gene microarray