Generation and performance of an equine-specific large-scale gene expression microarray.
Abstract: 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. Methods: Public nucleotide sequence database from Equus caballus and synoviocytes from clinically normal adult horses. Methods: 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: 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.
Publication Date: 2005-01-06 PubMed ID: 15631031DOI: 10.2460/ajvr.2004.65.1664Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article is about the creation and testing of an equine gene expression microarray, which was specifically designed to evaluate the effects of lipopolysaccharide exposure on horse synoviocytes.
Creation of the Equine Microarray
- The researchers began with a public database of gene sequences from Equus caballus (the horse) and synoviocytes (connective tissue cells) from healthy adult horses.
- A total of 18,924 equine gene sequences were initially used.
- Through computational procurement and annotation, the researchers selected 3,098 equine 3′ sequences that met the criteria for inclusion on the gene expression microarray.
- An oligomicroarray (a DNA microarray where probes are synthetically created oligonucleotides) was then manufactured. Probes were 25-oligomers, and a total of 68,266 were used to uniquely identify each gene.
Evaluation of the Microarray’s Performance
- The performance of the microarray was evaluated by observing the responses of equine synoviocytes to lipopolysaccharide (LPS), a molecule known to cause inflammation.
- Most genes on the microarray (68%) were expressed in equine synoviocytes. This high expression level denotes the suitability of the microarray for sequencing horse genes.
- The repeatability of the measurements was exceptionally high (r, > 0.99), which reveals that the microarray consistently gives reliable results.
- LPS caused significant changes, upregulating 84 genes (more than fivefold) and downregulating 14 genes (more than fivefold). Amongst the upregulated genes were many inflammatory mediators, shedding light on how LPS affects synoviocytes.
Conclusions and Significance of the Study
- The process used to curate the equine sequence database and create the microarray was successful in generating high-quality, species-specific gene sequences and probe sets.
- The microarray was effective at tracking changes in gene expression associated with LPS exposure in equine synoviocytes, making it a potentially valuable tool for understanding inflammation responses in horses.
- The research expanded the equine public database from 290 annotated genes to more than 3,000 provisionally annotated genes, greatly enhancing the available genetic knowledge for this species.
- The study suggests that similar curation and annotation of public databases could be used to create species-specific microarrays for other animals.
Cite This Article
APA
Gu W, Bertone AL.
(2005).
Generation and performance of an equine-specific large-scale gene expression microarray.
Am J Vet Res, 65(12), 1664-1673.
https://doi.org/10.2460/ajvr.2004.65.1664 Publication
Researcher Affiliations
- Comparative Orthopedic Molecular Medicine Research Laboratories, Department of Veterinary Clinical Science, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA.
MeSH Terms
- Animals
- Cells, Cultured
- Escherichia coli
- Gene Expression / physiology
- Gene Expression Profiling / veterinary
- Horses / genetics
- Lipopolysaccharides / pharmacology
- Oligonucleotide Array Sequence Analysis / veterinary
- RNA, Messenger / metabolism
- Synovial Membrane / cytology
- Synovial Membrane / drug effects
- Up-Regulation
Citations
This article has been cited 5 times.- Murray SJ, Santangelo KS, Bertone AL. Evaluation of early cellular influences of bone morphogenetic proteins 12 and 2 on equine superficial digital flexor tenocytes and bone marrow-derived mesenchymal stem cells in vitro. Am J Vet Res 2010 Jan;71(1):103-14.
- Barrey E, Mucher E, Jeansoule N, Larcher T, Guigand L, Herszberg B, Chaffaux S, Guérin G, Mata X, Benech P, Canale M, Alibert O, Maltere P, Gidrol X. Gene expression profiling in equine polysaccharide storage myopathy revealed inflammation, glycogenesis inhibition, hypoxia and mitochondrial dysfunctions. BMC Vet Res 2009 Aug 7;5:29.
- Yuan ZQ, Nicolson L, Marchetti B, Gault EA, Campo MS, Nasir L. Transcriptional changes induced by bovine papillomavirus type 1 in equine fibroblasts. J Virol 2008 Jul;82(13):6481-91.
- Chowdhary BP, Raudsepp T. The horse genome derby: racing from map to whole genome sequence. Chromosome Res 2008;16(1):109-27.
- Santangelo KS, Johnson AL, Ruppert AS, Bertone AL. Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells. Arthritis Res Ther 2007;9(1):R1.
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