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DJ Brown, SR Atilano, MC Kenney; Gene Expression Profiling In Human Age-related Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2834.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To assess alterations in retinal and pigmented epithelium (RPE) gene expression that accompany age related macular degeneration (AMD) in human patients. Methods:Retinal and RPE tissues were obtained from autopsy material of patients with documented histories (including fundus photos) of AMD (n=8). Age and sex matched control tissues were obtained through the National Disease Research Interchange. Total RNA was isolated by extraction using Trizol (Invitrogen) and Poly A+ RNA isolated from the total RNA by magnetic bead separation. RNA quality and quantity were evaluated using an Agilent Bioanalyzer. Samples were then processed using the Smart cDNA protocol and the resulting cDNA was labeled with 32P-dATP. This material was then hybridized to cDNA arrays containing 3600 unique elements. Images were obtained using a Molecular Dynamics phosphorimager and stored as 16 bit TIF files. These files were evaluated using the Imagene processing software (BioDiscovery) and the results analyzed using GeneSight software (BioDiscovery). Results:The RNA obtained from these postmortem human tissues was of a consistent quality and quantity to utilize as a source for array based expression profiling. Smart cDNA synthesis was found to be highly reproducible and gave results similar to those obtained by direct RNA labeling methods. In this survey of 3600 genes, several hundred genes were identified with a 99% confidence level as either over or under expressed in AMD. Conclusion:Human postmortem tissue contains sufficient levels and quality of RNA to reliably perform array based analyses for gene expression profiling. Our results suggest that AMD retinal and RPE tissues have a variety of changes that generally cluster into functional groups encompassing alterations in extracellular matrix, cytokine profile, plasma membrane receptors and cell cycle regulators.
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