Challenges of Human RPE Gene Profiling

Abstract

Abstract: : Purpose: To develop and refine methodologies for validating RPE cell–associated gene expression identified by Serial Analysis of Gene Expression (SAGE). Methods:A representation of overall gene expression in human neural retina and RPE/choroid was obtained using SAGE. RPE genes showed elevated expression (increased tag counts) in the RPE/choroid–derived libraries compared to the retina–derived libraries. By comparing these tissue–specific SAGE libraries, a number of potential RPE–associated genes were identified. In order to distinguish RPE–derived transcripts from choroidal expression, an RPE cell–enriched sample was prepared against which RPE/choroid gene expression was compared using real–time RT–PCR. Following removal of the retina from RNAlater–treated human posterior poles, RPE cells were mechanically enriched by gently brushing them away from Bruch’s Membrane. Further RPE cell purification was achieved by using flow cytometry and differential sedimentation. Results:Enrichment of RPE cells in our samples was verified by light microscopy and real–time RT–PCR, which showed higher relative expression of known RPE–specific genes (i.e. retinaldehyde binding protein 1) in the RPE–enriched samples than in the RPE/choroid samples. Similarly, RPE–associated candidate genes identified using SAGE were validated by comparing relative gene expression in RPE–enriched samples to RPE/choroid and neural retina samples. Conclusions:Validating expression of human RPE–associated candidate genes relies on the ability to collect highly enriched samples of RPE cells. Careful removal of RPE cells from Bruch’s Membrane using a brush followed by a cell density separation yields RPE cells devoid of any significant contamination from other retinal cells. The methodologies developed in this body of work serve as essential tools for identifying genes expressed in the RPE cell. Furthermore, by separating RPE cells based on their location relative to the adjacent retina and purifying them in the aforementioned manner, regional differences in RPE gene expression can also be elucidated.

Keywords: retinal pigment epithelium • candidate gene analysis • gene/expression