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A. Arora, D. A. C. Simpson; Retinal MicroRNA Prediction and Verification: A Novel Method Based on Target Gene Expression. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3776.
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MicroRNAs (miRNAs) are short (approximately 22nts) non-coding RNAs which regulate gene expression. Although they act principally at the post-transcriptional level they have also been shown to have some effect on mRNA levels. miRNAs have been detected in a range of tissues and shown to perform important tissue-specific and developmental functions. Very little is known about their expression in the retina. Therefore the purpose of this study was to establish how effectively a novel method, based on target gene expression, could predict the miRNAs expressed in the retina.
Retinal microarray gene expression datasets and lists of miRNAs, along with the genes they are predicted to target, were downloaded from Gene Expression Omnibus (NCBI GEO) and TargetScan respectively. miRNAs with predicted target genes expressed at a significantly lower than average level were selected. To confirm the expression of these miRNAs, human retinal RNA (BD Biosciences) and RNA extracted from rodent retina, was polyadenylated and reverse-transcribed with an oligodT linker primer (RACE kit, Invitrogen). Conventional and qPCR (Lightcyler, Roche) was performed using the reverse RACE primer and specific miRNA primers.
Six independent retinal microarray gene expression datasets were analysed. Significant evidence for 40 miRNAs (P<0.05) was detected, including known retinal and neural miRNAs. Signatures for 8 of these were detected in all 6 datasets, with another 8 miRNAs present in at least 4 datasets. The expression of candidate miRNAs, including miR-124a, miR-29 and let-7 was verified using RT-PCR.
In addition to their effects on protein expression miRNAs alter mRNA gene expression profiles, creating a signature which can be used to predict the miRNAs expressed in the respective tissues. Many miRNAs previously known to regulate gene expression in the brain and retina were successfully identified using this data mining approach. The confirmation of specific miRNAs expressed in the retina underlines the likely importance of these small RNAs in this tissue.
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