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FE Coblentz, MJ Radeke, SF Geller, GP Lewis, SK Fisher; Gene Array and QPCR Analysis Following Experimental Retinal Detachment . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4538.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To identify and characterize changes in gene expression in the retina following detachment. Methods:RNA was isolated from retinas that were experimentally detached for 1, 3, or 28 days, using cesium chloride precipitation. P33 labeled cDNA was made using the Ambion StripEZ kit and was then hybridized to a human "Named Genes" filter (Research Genetics) overnight at 37C. The filter was washed, exposed to a phosphoimaging screen, recorded using Molecular Dynamics software, and analysed using LabWorks software. Primer sets were subsequently designed for genes of interest and QPCR performed using RNA isolated from retinas detached for 0.25, 3, 7 and 28 days. Results:Of the 4,184 genes on the array, approximately 1,400 appeared to be in the retina. While expression levels of most genes did not change following detachment, several hundred did show a change of at least 1 SD from normal. For example, vimentin, GAP 43, transcription factor 12, and C-src kinase were dramatically upregulated and matrix metalloproteinase 2 was significantly downregulated. QPCR of these 5 genes supported these findings. Last year we reported that GAP 43 expression increased after detachment and that this increase was localized to a subpopulation of ganglion cells. Here we show by QPCR that GAP 43 mRNA increased 1.91, 1.72, 1.21, and 0.996 fold at 0.25, 3, 7 and 28 days after detachment, respectively. Although not on the arrays, we also performed QPCR on S- and M/L cone opsins and observed a significant down regulation. Conclusion:One of the problems with studying changes in gene expression after detachment has been deciding which genes to study. Gene array analysis and QPCR provide powerful tools for selecting and quantifying the changes in gene expression following detachment. Indeed, these techniques ultimately led to the identification of unsuspected reactivity, and plastic changes in ganglion cells. Of the 5 genes listed here, prior information had only indicated vimentin as a logical choice for study. The other 4 may provide new clues about the retina's reaction to detachment.
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