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L. Crosson, R. Kroes, J. Moskal, R.A. Linsenmeier; Effects of Hypoxia on mRNAs in the Rat Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2588.
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© ARVO (1962-2015); The Authors (2016-present)
Many blinding diseases involve tissue hypoxia. Very little is known about the molecular consequences of hypoxia in the retina beyond induced increases in VEGF mRNA expression. We therefore performed microarray analysis of the rat retina transcriptome under normoxic and hypoxic conditions to identify possible new targets for interrupting retinal disease.
Using pigmented rats, we investigated mRNA expression in the retina following hypoxia using a microarray platform and qRT–PCR. Hypoxia was induced by placing rats in a chamber in which the oxygen percentage was maintained at 6–7%O2 for 3 hr. Following hypoxia, animals were either sacrificed or allowed to recover in air for 24 hrs and then sacrificed. Retinas were removed. For microarrays, total RNA was extracted and aRNA was prepared. aRNA was also prepared from a universal rat reference sample. Both samples were labeled with Cy5 and Cy3 dye, respectively, combined, denatured and hybridized to arrays. Arrays were scanned and images were analyzed with BlueFuse (BlueGenome Ltd.). Data files were normalized using LOWESS curve–fitting equation. To identify statistically significant differentially expressed genes, analyses were performed with SAM. For qRT–PCR, cDNA was generated and amplified in the presence of SYBR® Green. Quantification of gene expression was determined by Ct cycle method.
From microarrays, 129 genes were upregulated during hypoxia and 121 genes were upregulated during recovery. 28 genes were downregulated during hypoxia and 29 genes during recovery. Of these genes, 92 genes were upregulated at both time points, and 13 were downregulated. Several genes involved in ion channel regulation and metabolism were identified as well as several genes relevant to vascular biology. Known HIF–1 target genes were also identified. Through qRT–PCR, several genes not present on the microarray, including VEGF and its receptors, and erythropoietin were also upregulated by hypoxia. mRNA differences were observed between controls and both hypoxic and recovery samples for three of the principal proteins in the phototransduction cascade of rods, including arrestin, calmodulin and the cyclic nucleotide gated channel. Similar expression trends were seen in microarray and qRT–PCR results for these genes.
Through both microarray technology and qRT–PCR, we have shown the regulation of several different gene families by hypoxia. In doing so, we have identified several different pathways which may be relevant to retinal disease progression. Supported by Falk Foundation
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