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U. Weitgasser, K. Oetsch; Gene Expression Profiling of the Response of Human Retinal Pigment Epithelial (RPE) Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1716.
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Purpose: Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells and pathological studies indicate that damage to the retinal pigment epithelium (RPE) is an early event in AMD. In vitro studies show that oxidant treated RPE cells undergo apoptosis, a possible mechanism by which RPE cells are lost during early phase of AMD. Methods: Two compounds that initiate cellular oxidative stress (Menadione and thiomalic acid) were used independently and in different concentrations and the effects on the two different cell types; human bronchial epithelial cells (HBEC) and RPE were compared. Trypan blue was used to determine the extent of cell viability and DNA fragmentation was detected by TUNEL staining. Poly- (A)+ mRNA was extracted with the FastTrack 2.0 kit (Invitrogen). Fluorescently labeled cDNA probes were prepared by incorporation of Cy3- and Cy5-conjugated dUTP during cDNA synthesis using a commercially available standard reference pool of mRNA (Stratagene universal reference) and the experimental sample of mRNA, respectively. Agilent microarrays were used (16,000 cDNA clones, 13,000 non-redundant genes). These gene expression profiles were compared by hierarchical cluster using TreeView algorithm. We confirmed several observations in an independent set of RPE samples by using quantitative RT-PCR. Results: Gene expression patterns clearly distinguished normal from apoptotic cells showing genes involved in several cellular functions being induced or repressed. Conclusions: Some of the genes identified as substantially induced or repressed may be relevant to AMD pathogenesis. Studies on cultured RPE cells can serve as a reference framework for future profiling of AMD tissues.
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