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David A. Simpson, Anna O' Connor, Jasenka Guduric-Fuchs, Tim M. Curtis; Characterisation of the Effect of miR-26a Upon Global mRNA Expression in Bovine Retinal Microvascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5133.
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© ARVO (1962-2015); The Authors (2016-present)
Primary bovine retinal microvascular endothelial cells (BRECs) constitute a widely-used model of angiogenesis. Information about mRNA expression is crucial to understand how these cells perform their roles in angiogenesis, barrier selectivity and control of vascular tone. However, because the cow is not a standard model organism, tools to measure global gene expression have been limited. The aim of this study was to apply only recently available ‘next generation’ sequencing techniques to characterise the mRNA profile of BRECs. Furthermore, we wanted to measure the effect of altering a specific microRNA upon the mRNA profile.
RNA was isolated from primary cultures of untreated BRECs or following transfection with an LNA to inhibit, or a plasmid to over-express, miR-26a. cDNA libraries were prepared using a ScriptSeq kit (Epicentre) and sequenced on an Genome Analyzer (Illumina). Reads were mapped to the bovine genome (UMD3.1) using Genomics Workbench software (CLCbio) and the number of reads mapped per kilobase of each gene, per million fragments mapped (FPKM) calculated.
Approximately 12 million reads were obtained from each library, of which ~8 million mapped to the genome. Extremely highly expressed mRNAs previously associated with endothelial cells and angiogenesis included Thrombospondin 1 (820 FPKM) and Thymosin beta10 (1060 FKM). Modulation of miR-26a expression caused significant changes in mRNA expression. Altered genes included predicted miR-26a targets such as PECAM1 and many potential novel targets including Angiopoietin-2 and Endothelin-1. Mapping of reads to exons facilitated identification of splicing patterns and many examples of novel alternatively spliced transcripts were observed.
RNA-Seq enables the quantitative evaluation of mRNA expression and splicing patterns without prior sequence knowledge, an advantage over hybridization-based array approaches, particularly in less well characterized organisms. This global profile of BREC mRNA expression will be an invaluable resource for future studies of endothelial biology. The significant mRNA changes observed following manipulation of miR-26a underline the important role of microRNAs in regulating endothelial gene expression.
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