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R. Deniskin, A. Yuan, S. Kar, E. L. Farber, N. B. Akhmedov, D. B. Farber; MicroRNA Expression in the Retinal Degeneration (rd1) Mouse Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5781. doi: https://doi.org/.
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MicroRNAs are small (~22 nucleotide) non-coding RNAs that act as translational repressors via their complementation with the 3'UTR region of target mRNA sequences. They play a significant role in developmental patterning, apoptosis, and tissue differentiation. This study examines the global miRNA expression profile in the retinal degeneration ( rd1) mouse model.
We profiled the expression of retinal miRNAs in wildtype C57Bl/6 and rd1 mice using microarray technology. Differences in miRNA levels were further confirmed by quantitative real-time PCR. Total RNA was reverse-transcribed using miRNA-specific RT primers. RT products were amplified in triplicates by TaqMan qPCR and quantified using a semi-quantitative comparative Ct method. Statistical differences were determined using bootstrap statistics to simulate a two-tail student t-test and one-way analysis of variance (ANOVA). Differences with P< 0.05 were considered statistically significant.
Microarray patterns for the top 10% of the most highly expressed miRNAs were statistically analyzed and validated by qPCR. The levels of miR-16, a ubiquitously expressed "housekeeping" miRNA, showed two-to-four fold decrease in qPCR in the rd1 versus wild type C57BL/6, which was consistent with the microarray data. The most robust decreases were observed in the miRNA clusters of -96 and -183, showing 5-6-fold and four-fold decreases, respectively. Interestingly, miR-182 which is part of the same miR-183 cluster showed a decrease by at least one order of magnitude. This cluster has been predicted to target MITF (micropthalmic transcription factor) which has been shown to be required for the development and maintenance of the RPE. In addition, the level of miR-93, a member of the miR-17 family cluster, showed a 2-3 fold decrease in the disease mouse compared to the wildtype control. Furthermore, miR-let7f, which was examined as a no-change control, showed no difference between the tested mice in both the microarrays and qPCR experiments, as expected. Our results also provide convincing evidence for higher expression levels for certain miRNAs which showed at least two-fold increases in rd1 mice.
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