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Michael R. Frost, Lin Guo, Thomas T. Norton; Whole Transcriptome Analysis of Tree Shrew Sclera during the Development of Lens-induced Myopia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3452.
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During negative lens-induced myopia there is a regulated remodeling of the scleral extracellular matrix (ECM). Whole transcriptome analysis (RNA-Seq) was used to identify novel genes that are differentially expressed during this process. A comparison was made with quantitative real-time PCR (qPCR), the current ‘gold standard’ method for assessing differential gene expression.
To induce myopia, 7 tree shrews wore a monocular –5 D lens for 4 days, starting 24 days after eye opening. RNA-Seq was used to measure the relative differential change in mRNA levels for the whole transcriptome using RNA from one of these animals. RNA-Seq was performed on a HiSeq2000 platform using a TruSeq library generation kit (Illumina). RNA-Seq reads were aligned to the tree shrew genome using TopHat/Bowtie and then transcripts were assembled with Cufflinks, followed by analysis with Cuffdiff to identify significant changes in transcript expression. For comparison/validation, mRNA abundance changes for 38 candidate genes present in the RNA-Seq dataset were also assessed with qPCR, in all 7 animals.
Negative lens treatment induced a –3.0 ± 0.3 D myopic shift (mean ± SEM) relative to the untreated fellow control eyes (–2.8 D for the individual RNA-Seq animal). RNA-Seq identified 20,820 distinct transcripts, of which 2,109 were significantly differentially expressed in response to lens treatment. Overall, there was a highly significant correlation for the direction and magnitude of the differential fold changes for the 38 candidates between the two methods (see figure). Several novel functional classes of genes were identified as being regulated by negative lens wear, the most prominent of these being genes involved in intercellular signaling and ion transportation. In addition, RNA-Seq confirmed the previously reported involvement of genes associated with ECM and cytoskeleton remodeling, cell adhesion, transcription, and general cellular growth.
RNA-Seq can provide simultaneous discovery and quantification of novel genes with altered expression in tree shrew sclera, unlike microarrays, as well as confirmation of genes known to change in abundance. This represents a paradigm shift from the usual candidate gene approach.
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