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Michael H. Farkas, Gregory R. Grant, Brian Cole, Eric A. Pierce; Comprehensive Characterization Of The Wild-type Mouse Neural Retina Transcriptome Using Next-generation Sequencing. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2560.
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Using RNA-seq, we have extensively characterized the mouse neural retina transcriptome by identifying and validating novel alternative splicing events. It is essential to have a completely annotated transcriptome of a wild-type control tissue for analysis of various retinal diseases. For example, it has been hypothesized that the mechanism of disease pathogenesis for the RNA splicing factor forms of RP involves the production of aberrantly-spliced transcripts. With an updated transcriptome, it will be possible to differentiate between transcripts that are aberrantly-spliced, relative to those that are novel, non-pathogenic, alternative transcripts.
RNA-seq libraries were prepared from total retinal RNA isolated from two month old wild-type C57BL/6J mice. The libraries were sequenced using 120bp paired end reads on an Illumina Genome Analyzer IIx. Sequence reads were aligned to the mouse genome and analyzed using the RNA-seq Unified Mapper (RUM), a pipeline for RNA-seq data analysis. The relative levels of gene expression and selected novel alternative splicing events were validated by RT-PCR and Sanger sequencing.
Over 20% of the 100,000,000 reads sequenced per RNA-seq library were found to cross exon junctions. The majority of these reads crossed 125,000 annotated junctions, while the remaining reads crossed 47,000 novel junctions at >2 read depth. Over 12,000 of these junctions identified novel alternative splicing events, including exon skipping, new exons, and alternate splice sites. We selected and validated 25 novel events from each of these categories. Twenty one/fifteen exon skipping, 19/9 novel exons and 19/14 alternate splice sites were validated by RT-PCR and sequencing, respectively.
These transcriptome analyses demonstrate that the current understanding of the gene expression and transcript variation in the neural retina is incomplete. This is an important finding because a comprehensively annotated transcriptome is important for investigating and understanding many aspects of retinal biology and disease. For example, identification of novel tissue-specific alternative splicing events can inform genetic analyses of retinal disorders and investigations of disease pathogenesis.
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