Generation and performance of an equine-specific large-scale gene expression microarray

Weisong Gu Comparative Orthopedic Molecular Medicine Research Laboratories, Department of Veterinary Clinical Science, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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Alicia L. Bertone Comparative Orthopedic Molecular Medicine Research Laboratories, Department of Veterinary Clinical Science, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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Abstract

Objective—To create high-quality sequence data for the generation of an equine gene expression microarray and evaluate array performance by use of lipopolysaccharide (LPS) exposure of synoviocytes.

Sample Population—Public nucleotide sequence database from Equus caballus and synoviocytes from clinically normal adult horses.

Procedure—Computer procurement of equine gene sequences, probe design, and manufacture of an oligomicroarray were performed. Array performance was evaluated by use of patterns for equine synoviocytes in response to LPS.

Results—Starting with 18,924 equine gene sequences, 3,098 equine 3' sequences were annotated and met the inclusion criteria for an expression microarray. An equine oligonucleotide expression microarray was created by use of 68,266 of the 25-oligomer probes to uniquely identify each gene. Most genes in the array (68%) were expressed in equine synoviocytes. Repeatability of the array was high (r, > 0.99), and LPS upregulated (> 5-fold change) 84 genes, many of which were inflammatory mediators, and downregulated (> 5-fold change) 14 genes. An initial pattern of gene expression for effects of LPS on synoviocytes consisted of 102 genes.

Conclusions and Clinical Relevance—Use of a computer algorithm to curate an equine sequence database generated high-quality annotated species-specific gene sequences and probe sets for a gene expression oligomicroarray, which was used to document changes in gene expression associated with LPS exposure of equine synoviocytes. The equine public database was expanded from 290 annotated genes to > 3,000 provisionally annotated genes. Similar curation and annotation of public databases could be used to create other species-specific microarrays. (Am J Vet Res 2004;65:1664–1673)

Abstract

Objective—To create high-quality sequence data for the generation of an equine gene expression microarray and evaluate array performance by use of lipopolysaccharide (LPS) exposure of synoviocytes.

Sample Population—Public nucleotide sequence database from Equus caballus and synoviocytes from clinically normal adult horses.

Procedure—Computer procurement of equine gene sequences, probe design, and manufacture of an oligomicroarray were performed. Array performance was evaluated by use of patterns for equine synoviocytes in response to LPS.

Results—Starting with 18,924 equine gene sequences, 3,098 equine 3' sequences were annotated and met the inclusion criteria for an expression microarray. An equine oligonucleotide expression microarray was created by use of 68,266 of the 25-oligomer probes to uniquely identify each gene. Most genes in the array (68%) were expressed in equine synoviocytes. Repeatability of the array was high (r, > 0.99), and LPS upregulated (> 5-fold change) 84 genes, many of which were inflammatory mediators, and downregulated (> 5-fold change) 14 genes. An initial pattern of gene expression for effects of LPS on synoviocytes consisted of 102 genes.

Conclusions and Clinical Relevance—Use of a computer algorithm to curate an equine sequence database generated high-quality annotated species-specific gene sequences and probe sets for a gene expression oligomicroarray, which was used to document changes in gene expression associated with LPS exposure of equine synoviocytes. The equine public database was expanded from 290 annotated genes to > 3,000 provisionally annotated genes. Similar curation and annotation of public databases could be used to create other species-specific microarrays. (Am J Vet Res 2004;65:1664–1673)

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