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Anna O'Connor, Jasenka Guduric-Fuchs, Elayappan Banumathi, Angela Cullen, Timothy M. Curtis, David A. Simpson; Deep Sequencing Reveals Predominant Expression of a Small Number of MicroRNAs in Bovine Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1422.
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MicroRNAs are small, endogenous, non-coding RNAs which regulate mRNA expression at the post-transcriptional level. The crucial functions of retinal vascular endothelial cells in angiogenesis, barrier selectivity and control of vascular tone rely upon appropriate regulation of gene expression. Although microRNAs undoubtedly contribute to the regulatory process, those present in endothelial cells are poorly characterised. The aim of this study was to identify and quantify microRNAs expressed in bovine retinal endothelial cells (BRECs) using next generation sequencing.
BRECs were isolated and RNA extracted from passage 2 cultured cells. A small RNA library was prepared and deep sequencing performed (Illumina Genome analyser). Reads were trimmed and mapped to known microRNAs in miRBase (release 16) using Genomics Workbench software (CLCbio).
After removal of linker sequences from 20 million raw reads, approximately 10 million 15-25nt reads potentially representing microRNAs remained. Of these, 6.8 million reads mapped to known bovine microRNAs. More than 70 novel bovine orthologs of known human microRNAs were also detected and additional putative novel microRNAs were identified from reads mapped to stem loop precursors. Heterogeneity was observed in microRNA length particularly at the 3' end. Highly expressed microRNAs previously associated with endothelial cells and angiogenesis included miR-126 (1,014,272 mapped reads) and miR-378 (302,311 mapped reads). Approximately 75% of all mapped reads were derived from the 5 most highly expressed microRNAs: let-7a, let-7f, miR-21, miR-126, and miR-378. The expression of candidates with a range of reads was confirmed by qRT-PCR.
The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in BRECs. Characterisation of the functions of these highly expressed microRNAs will provide insights into endothelial biology.
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