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T.L. Young, G. Scavello, J. Choi, E. Rappaport; Human Sclera Gene Expression Profile Using cDNA Microarray Analysis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):411.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop gene expression profiles of human sclera to allow for the identification of novel, uncharacterized genes, and to identify candidate genes for scleral disorders. Methods: Total RNA was isolated from 6 donor sources of human sclerae, and reverse transcribed into cDNA using a T7-(dT)24 primer. Resulting cDNA was in vitro transcribed to produce biotin-labeled cRNA, fragmented, and mixed with hybridization controls before a 16-hour incubation/ hybridization step to oligonucleotide probes on 6 Affymetrix U95A chips. The chips were scanned twice at 570 nM and the data collected using GeneChip software. Array analyses were carried out with Microarray Suite, version 5.0 (Affymetrix), using the expression analysis algorithm to run an absolute analysis after cell intensities were computed. All arrays were normalized to the same target intensity using all probe sets. Reverse- transcription PCR was performed to validate the microarray results. Results: Labelled, fragmented scleral cRNA hybridized to more than 58 % of the 12,626 probe sets represented on the microarray chip. There were 3769 genes with "present" calls assigned independently to all six human scleral samples. These genes could be clustered into 4 major categories: transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). Many extracellular matrix proteins, such as collagens 6A3 and 10A1, thrombospondins 2 and 4, versican, and dystroglycan have not previously been shown to be expressed in sclera. RT-PCR results confirmed expression in 6 of 6 genes examined. Conclusions: This study demonstrates the utility of gene microarray technology in identifying global patterns of scleral gene expression, and provides the first comprehensive list of genes expressed in human sclera. Identification of genes expressed preferentially or exclusively in sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.
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