Microarray Study of the Aging mRNA Phenotype of the Mouse RPE/Choroid

Abstract

Abstract: : Purpose: This study was undertaken to investigate age-related change of the mRNA phenotype in the mouse RPE/choroid. The study design using replicates of individual mouse samples and the use of an inbred strain allowed statistical analysis of our data which in turn yielded a very high level of confidence in our results. Methods:C57Bl6 mice at either 6 weeks or 24 months were sacrificed and both globes from each individual mouse were processed to extract total RNA from the RPE/choroid. RNA was then amplified using the Clontech SMART system and 3 individual samples of 6 week old and 24 month old animals were each labeled and used to probe the Clontech 1.2 and 1.2 II mouse arrays (a total of 12 blots). Data were collected on a phosphorimager and normalized using software. Statistical analysis was performed using the SAM (Statistical Analysis of Microarray) software from Stanford University. Results:A large number of genes (156) were differentially expressed between young and old mouse RPE/choroid samples. These were grouped into several functional categories (inflammatory response, lysosomal proteases, and oxidative stress response, etc.). The false discovery rate (FDR) for all 156 genes was less than 7% and many of these genes (90) were less than 2%, reflecting the quality of the data set. Upon survey, several candidate genes including cathepsin S, stromelysin, and glutathione peroxidase 3 were selected for study with respect to the cell biological pathways which may contribute to the development of the changes in Bruch’s membrane in the aging mouse eye. Changes in the RNA levels of these transcripts were verified by PCR and in situ hybridization. Conclusions:Further analysis of the promoters of the cathepsin S and stromelysin genes indicate that these may be oxidative stress genes. This finding may point towards a hypothesis that a substantial part of the aging phenotype of the RPE/choroid is very similar to an oxidative stress phenotype.

Keywords: gene microarray • retinal pigment epithelium • gene/expression