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K. Stasi, J. Danias, X. Yang, S.M. Podos, T. Mittag; Differential Gene Expression in Glaucomatous DBA/2 Mouse Retinas . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1242.
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Purpose: To determine genes that are up– or down–regulated in the retina of glaucomatous DBA/2 mice with the progression of the disease by comparing microarray(MA), RT–PCR and Western blot (WB) analyses. Methods: RNA was extracted from retinas of 3 and 15 month old DBA/2 glaucomatous mice and subjected to MA gene analysis (Affymetrix). Differential gene expression was determined by several statistical methods (MAS5.0, SAM, RMA analysis, and B–H procedure). RT–PCR analysis of seven genes of interest, in comparison to the housekeeping gene rps, was performed using pooled RNA from 6 retinas for each of 5 age groups (3, 6, 9, 12 and 15 months old) of DBA/2 and non–glaucomatous C57/BL6 mice. Two genes with high differences between the two mouse strains in MA and RT–PCR were also confirmed by WB analysis. Results: Microarray data indicated 33 genes were significantly up–regulated in glaucomatous DBA/2 retinas compared to pre–pathologic ones, with a false positive rate of less than 1. Complement component 1 q (C1q) levels in the aged glaucomatous DBA/2 retina were elevated 11–fold, 8–fold and 30–fold compared to the levels in pre–pathologic retina when measured by MA, RT–PCR and WB respectively. C1q levels in retina of non–glaucomatous aged C57/BL6 mice increased 2–fold by RT–PCR but were unchanged by WB compared to levels in younger animals. Ceruloplasmin (Cp) levels in the aged DBA/2 retina were elevated 4–fold (MA), 15–fold (RT–PCR), and 4–fold (WB). Cp levels in aged C57/BL6 retina increased 3–fold (RT–PCR) while they were unchanged by WB. C1q and Cp were 2 of the 4 genes that were consistently increased more than 3–fold by the 9th month of age. Osteopontin mRNA was up–regulated 1.5 fold by MA and 2–fold by RT–PCR in the aged DBA/2 retina. Neurofilament–200, 1 of 5 genes down–regulated by MA (0.7 fold) was found to be 1.5 fold up–regulated by RT–PCR. Thy–1, PGP 9.5 and matrix metalloproteinase 9 (MMP 9), 3 genes associated with retinal ganglion cells, were unchanged by MA analysis. Thy 1 and PGP 9.5 also showed no change by RT–PCR. MMP–9 levels were up–regulated 2–fold in aged glaucomatous DBA/2 retinas by RT–PCR. Conclusions: Microarray analysis of gene expression is a powerful tool in the study of the molecular mechanisms of the retinal stress response to factors such as elevated intraocular pressure, and in the pathogenesis of glaucoma. Detected changes in gene expression may depend on the method used to analyze microarray data and require confirmation by an independent method (e.g. RT–PCR and/or WB).
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