Whole retinal transcriptome analysis of early stages of AMD

Abstract

Abstract: : Purpose: Age–related macular degeneration (AMD) affects predominantly photoreceptor cells and the retinal pigment epithelium (RPE). The aim of this study was to identify changes in gene–expression related to early stages of AMD in RPE cells. In late stages of AMD the central retina is degenerated, which prohibits gene–expression analysis. Methods: We defined early stages of AMD in human post–mortem eyes and isolated RPE cells using laser dissection microscopy. Next, an oligonucleotide array enriched in genes expressed in retinal tissue was developed. Human donor eyes were obtained from the Dutch Cornea Bank. Cryosections were cut from the macular area. Ten sections (200 µM apart) were PAS–stained and evaluated for morphological quality, the presence of drusen or overt ocular pathology. RPE was isolated from unstained and unfixed sections using a laser dissection microscope. Next, RNA was isolated from the dissected cells. A custom oligonucleotide array (Agilent technologies) was designed by combining expression data from publicly available online libraries with expression data from human tissues (retina, RPE/Choroid, brain). This resulted in an array containing approximately 13.000 known and 9000 unknown genes detected in retinal tissue. Results:Drusen could be detected in cryosections after PAS staining and LM. Fourty eyes from donors (>70 yrs) were evaluated which resulted in the identification of 23% unaffected eyes (not a single drusen observed), 34% with a few drusen (<5) and 6% with many (>20) to numerous drusen which we qualified as early stage AMD. Four unaffected and 4 early stage AMD eyes were selected for RPE isolation. Of each eye 10.000 RPE cells were dissected and used for RNA isolation and amplification. Successful labelling and hybridisation with this material was carried out, demonstrating the feasibility of this approach towards the identification of early changes in gene–expression leading to AMD. Conclusion: Human post–mortem eyes available through donorbanks are valuable material especially in the analysis of degenerative eye diseases, like AMD. We demonstrate the possibility to use this material in a high throughput gene–expression profiling approach of early stages of AMD.

Keywords: age–related macular degeneration • drusen • gene microarray