Microarray analysis of gene expression in human donor sclera

摘要

Purpose: To develop gene expression profiles of human sclera toallow for the identification of novel, uncharacterized genes in thistissue-type, and to identify candidate genes for scleral disorders.Methods: Total RNA was isolated from 6 donor sources of humansclerae, and reverse transcribed into cDNA using a T7-(dT) 24 primer.The resulting cDNA was in vitro transcribed to produce biotin-labeledcRNA, fragmented, and mixed with hybridization controls before a 16 hhybridization step with oligonucleotide probes on 6 Affymetrix U95Achips. The chips were scanned twice at 570 nM and the data collectedusing GeneChip software. Array analyses were carried out with MicroarraySuite, version 5.0 (Affymetrix), using the expression analysis algorithmto run an absolute analysis after cell intensities were computed. Allarrays were scaled to the same target intensity using all probe sets.Reverse-transcription polymerase chain reaction (RT-PCR) was performedto validate the microarray results.Results: There were 3,751 genes with "present" calls assignedindependently to all six human scleral samples. These genes could beclustered 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 and10A1, thrombospondins 2 and 4, and dystroglycan have not previously beenshown to be expressed in sclera. RT-PCR results confirmed scleralexpression in 7 extracellular matrix genes examined.Conclusions: This study demonstrated the utility of gene microarraytechnology in identifying global patterns of scleral gene expression,and provides an extended list of genes expressed in human sclera.Identification of genes expressed in sclera contributes to ourunderstanding of scleral biology, and potentially provides positionalcandidate genes for scleral disorders such as high myopia.