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Michelle Dianne Drewry, Inas Helwa, R. Rand Allingham, Michael A Hauser, Yutao Liu; miRNA-Seq in four different normal human ocular tissues. Invest. Ophthalmol. Vis. Sci. 2016;57(12):794.
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MicroRNAs (miRNAs) are small, noncoding strands of RNA that negatively regulate post-transcriptional gene expression. Since miRNAs have been associated with many eye diseases, many studies have tried to profile the mammalian ocular miRNAs using microarray and realtime PCR. Our study aims to profile ocular miRNA expression in normal human eye tissue using miRNA-Seq.
Total RNAs were extracted from normal human ciliary body (CB) (n=2), cornea (n=2), retina (n=2), and trabecular meshwork (TM) (n=4) samples using mirVana total RNA isolation kit from Life Technologies. We used 1 µg RNA from each sample to prepare sequencing library with Illumina TruSeq Small RNA Library Prep kit with sample-specific indexes. Pooled libraries were sequenced using Illumina MiSeq. Generated sequence reads were trimmed and aligned against human reference database using Bowtie software. Only exact matches to known mature miRNA sequences were counted. The resulting miRNA profile was analyzed and compared using Microsoft Excel and the R Language and Environment for Statistical Computing. Relative expression level of miR-182 and miR-184 in these tissues was validated using realtime PCR and droplet digital PCR (ddPCR).
We identified 423 unique miRNAs in all 10 samples. We found that miR-143, miR-184, miR-204, miR-182, and miR-26a were the most abundant in all the examined ocular tissue. In CB and TM, miR-143 was the most abundant while miR-184 and miR-182 were the most abundant in cornea and retina, respectively. Many miRNAs were uniquely expressed in one or two tissues only. By analyzing the gene targets of the identified miRNAs, we were able to isolate a cluster of three miRNAs, expressed only in the cornea and TM, which target a common set of genes. Using the expression patterns, we were also able to create an expression profile of the miRNAs known to target genes associated with keratoconus and glaucoma. The relative expression of miR-182 and miR-184 in all examined tissues was validated using realtime PCR and ddPCR.
For the first time, we have profiled miRNA expression in multiple human ocular tissues using miRNA-Seq. We identified many miRNAs that had not been previously reported in ocular tissue. Knowing the expression of miRNAs in non-diseased eye tissues could help elucidate the miRNA profile change that accompanies diseases such as glaucoma and keratoconus.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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