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Philip Ruzycki, Matthew Brooks, Harsha Rajasimha, Sami Dridi, Hiroki Kaneko, Bradley Gelfand, Valeria Tarallo, Judit Baffi, Jayakrishna Ambati, Anand Swaroop; Differential miRNA Expression In Geographic Atrophy Retina And Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3918.
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Changes in gene expression have been reported as a consequence of aging and may contribute to the development of many aging-associated diseases. miRNA mediated gene regulation is thus of particular interest in the genesis of age-related macular degeneration. To test the hypothesis that the development of geographic atrophy, the advanced form of atrophic macular degeneration, is associated with differential miRNA expression, we utilized next generation sequencing to determine global profiles of miRNA expression in retinal pigment epithelium (RPE) and neural retina samples from 4 age matched control and 4 affected individuals.
Total RNA was purified from isolated human retina and RPE. Small RNA was apportioned by flashPAGE. Libraries were prepared using the Illumina small RNA v1.5 protocol and size selected for adaptor ligated 22bp miRNA. Completed libraries were loaded on the Illumina Cluster Station and sequenced on the Illumina GAIIx with 36 cycles of base incorporation. Image analysis and base calling was performed using the Illumina Pipeline. Reads were trimmed of adaptor sequences and all 15-35 bp sequences were mapped to the human genome and miRbase. Biological replicates were pooled and tag counts assessed for each miRNA.
The sequence data yielded about 82 million reads for affected and control groups. These reads have a median length of 22 bp and more than 82% map to miRbase. Preliminary analysis of retinal samples indicates a general decline in miRNA levels in affected individuals. Multiple miRNAs that determine translation of proteins involved in mitochondrial function, fibroblast response, and cell survival exhibited more than 2 fold decrease in GA patients compared to controls. This data will be validated by ongoing miRNA analysis of RPE samples and by complementary methods.
Differential miRNA abundance measured in the retina and RPE of geographic atrophy patients may contribute to the development of age-related macular degeneration.
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