Development of a microarray chip for gene expression in rabbitocular research

[摘要] Purpose: To develop a microarray for the rabbit that can be used forocular gene expression research.Methods: Messenger RNA was isolated from anterior segment tissues(cornea, conjunctiva, and iris) and posterior segment tissues (lens,retina, and sclera) of rabbit eyes and used to create two independentcDNA libraries through the NEIBank project. Clones from each of theselibraries were sequenced from both the 5' and 3' ends. These sequencesand those from the National Center for Biotechnology Information (NCBI)taxonomy database for rabbit were combined and electronically assembledinto a set of unique nonoverlapping continuous sequences (contigs). Foreach contig, a homology search was performed using BLASTX and BLASTNagainst both the NCBI NR and NT databases to provide gene annotation.Unique contigs were sent to Agilent Technologies, where 60 baseoligonucleotide probes were designed and synthesized, in situ, on twodifferent arrays in an 8 array x 1900 element format. Glaucomafiltration surgery was performed on one eye of six rabbits. After 14days, tissue was harvested from the conjunctiva and Tenon's capsule ofboth the surgically treated and untreated control eyes. Total RNA fromeach sample was labeled with cyanine dyes and hybridized to our custommicroarrays.Results: Of the 3,154 total probes present on the two arrays, 2,522had a signal value above the background. The expression of 315 genes wassignificantly altered by glaucoma filtration surgery. Genes whoseexpression was altered included proteins associated with inflammatoryresponse, defense response, and proteins involved in synthesis of theextracellular matrix.Conclusions: The results of this rabbit microarray study areconsistent with those from other wound healing studies, indicating thatthis array can provide valid information on broad patterns of geneexpression. This is the first microarray available for rabbit studiesand is a valuable tool that can be used to study molecular events in theeye.

[学科分类] 生物化学/生物物理