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K. Ishibashi, J. Tian, J. Handa; A Comparative Microarray Analysis of Morphologically Normal RPE Cells Laser Captured Microdissected from the Macula and Periphery of Human Donor Globes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2292.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the expression profiles of morphologically normal RPE cells overlying nonthickened Bruch’s membrane from the macula and periphery of human donor globes. Methods: Morphologically normal RPE cells overlying nonthickened Bruch’s membrane (n=5000 cells) from 10 donor eyes (ages 71-83 years) were laser capture microdissected. The RNA was extracted by column purification, and first strand cDNA probes were amplified using Super SMART. Probes were radiolabeled with 33P-dATP and hybridized to ResGen’s known human membrane array (n=4325 genes). The signal was visualized with phosphorimager analysis. The TIFF image was analyzed by ResGen’s Pathways 3 software. The data were normalized to a mean signal reference array, and analyzed using Cluster/Treeview and SAM. Real time PCR was used to confirm differential expression of selected genes. Results: An average of 95% of genes on the array were expressed. 114 of 165 genes (69%) were found expressed by macular laser captured RPE cells on this array that were identified from a recent RPE specific library. Cluster analysis in 6 of 10 eyes showed that the expression signature of macular and peripheral cells clustered by donor. No significant clustering by topographical origin of RPE cells was identified. Using SAM analysis with a 10% false discovery rate, 10 genes were identified as differentially expressed between macular and peripheral RPE. These genes were related to cell cycle regulation (n=5), metabolism (n=3), cell differentiation (n=1), and apoptosis (n=1). Conclusions: Microarray analysis of this set of globes suggests that the gene expression profile between normal macular and peripheral RPE cells is similar and most related to the specific donor. Differences in genes important to the general maintenance of the cell may influence phenotypic differences between macular and peripheral cells.
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