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Mariam S. Assadian, Verity F. Oliver, Kieron Torres, Ray A. Enke, Shannath L. Merbs; Retina-specific Dna Methylation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5984.
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DNA methylation is known to play a critical role in the differentiation of cells and tissues, and changes in DNA methylation have been shown to be important in the development of some diseases. We have previously demonstrated that certain retina-specific genes, including Rho and Rbp3, are differentially hypomethylated in the retina when compared to non-expressing tissues in the mouse. We believe that a genome-wide DNA methylation map of the retina will be invaluable as we search to understand the role of DNA methylation in retinal development and disease.
Whole retinas were extruded from 3 C57B/6J mice at 2 months of age. Mouse cortex was isolated as non-retinal control tissue. Genomic DNA was isolated and fragmented to an average of 300 bp. The fragmented DNA was divided in to 2 fractions: input DNA and a second fraction that was enriched for methylated DNA using MethylCollector (ActiveMotif). Enrichment was confirmed by QPCR of Rho, which is relatively unmethylated in the retina and methylated in the brain. The input DNA and methylated fractions were subjected to whole genome amplification (WGA), and maintenance of the enrichment was confirmed by QPCR, prior to being labeled and hybridized to mouse CHARM arrays (Comprehensive High-throughput Analysis of Relative Methylation, NimbleGen).
Multiple techniques for WGA were compared to insure that we were using the technique that best maintained our methylated DNA enrichment. The enriched and labeled DNAs are currently being evaluated in our ongoing, genome-wide analysis using the CHARM array. Retina-specific DNA methylation sites (when compared to brain) will be compared to known tissue-differentially methylated regions that have previously been identified by CHARM in other tissues.
Our pangenomic characterization of retina-specific DNA patterns provides an important resource for future studies to investigate the relationship between DNA methylation and retinal development and disease.
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