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G.L. Paez, B. Zangerl, G.M. Acland, G.D. Aguirre; Abnormal Gene Expression Profile in Retinas With RPGR Frame Shift Mutation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4897.
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© ARVO (1962-2015); The Authors (2016-present)
We have developed a custom canine retinal cDNA microarray containing ∼4,500 non–redundant elements from a normalized canine retinal cDNA library. To examine the gene expression profile of retinas with inherited photoreceptor degeneration, we have compared the expression profiles of normal and mutant retinas using a pooled brain RNA as a reference tissue; the affected retinas had a frameshift mutation in RPGR ORF15.
We employed a common reference sample–based experimental design in which mutant and normal retinas at the age of 3, 7 and 16 wks were hybridized in replicates against a reference sample of pooled brain RNA. Tissues were fluorescent labeled using an indirect labeling (3DNA Array 50TM kit). Data from normalized scanned images were analyzed using GeneSpring version 7.2 and SAM version 1.14 with a FDR of 2%. Northern blot analysis was used to confirm expression patterns.
Principal Component Analysis (PCA) showed a significant separation between each retinal group. Significance Analysis of Microarrays (SAM) analysis on pooled samples from affected retina identified 127 genes that were up– or down–regulated compared to normal retina. 98 genes were up regulated in the affected retina at the age of 16 weeks. Comparisons between affected retinas revealed that 13 genes were up– or down–regulated between 7–16 weeks, and only 11 genes showed change in expression patterns between 3–7 weeks.
The clustering of gene expression profiles in affected retinas at different ages of disease allows us to establish a developmental expression profile for this tissue using our microarray system. We identified retinal genes that are differentially regulated during the development and progression of photoreceptor degeneration. Our aim is to identify those that directly affect processes such as survival, apoptosis, and transcription as these are likely to play a pivotal role in the regulation of degeneration.
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