Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage.
Abstract: No large scale equine microarray is available commercially to allow genomic and transcriptional profiling of the majority of genes that would define the genetic basis of equine disease. Objective: To generate a whole transcript target labelled GeneChip to interrogate the equine transcriptome and validate chip performance using RNA samples derived from organs, articular cells and normal cartilage. Methods: Equine mRNA and selected equine gene sequences derived from perfect cross-hybridisation of equine RNA on human microarray GeneChips, were used to design a custom equine gene microarray. Sequence data were used as a template for generation of a glass-slide based 5'-3' multi-exon-encompassing gene chip. The microarray was characterised using RNA derived from organs including spleen, liver, brain and kidney, and RNA from cultured chondrocytes, cartilage, synovial tissue and stem cells, employing a whole transcript target labelling assay to sample mRNA across the 5'-3' spectrum. Results: The custom microarray simultaneously interrogated over 12,300 equine specific genes. Probing the chip with mixtures of total RNA derived from parenchymatous organs and articular tissues resulted in 61.7 and 62.8% present calls, respectively. This gene chip provided expression information on up to 90% of the key molecules in important signalling, metabolic and development pathways. Cartilage specific matrix genes were abundantly expressed in normal articular cartilage, but surprisingly high levels of collagen types I, III, V and XI, reflected expression from the epiphyseal layers of maturing articular epiphyseal cartilage. Conclusions: An oligonucleotide microarray with over 12,300 probe sets was generated by uniquely combining a labelling strategy incorporating expressed sequence tags from the entire transcriptome and supplementing selected human sequences that cross-hybridised with the horse. Validation showed robust performance of the microarray. Conclusions: This array may be a useful tool to elucidate the pathogenesis of equine diseases.
Publication Date: 2009-11-26 PubMed ID: 19927585DOI: 10.2746/042516409x412381Google Scholar: Lookup
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- Journal Article
Summary
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The research presents the creation of a novel equine-specific GeneChip microarray that can help identify the genetic factors driving equine diseases. It was tested using RNA from various organs and articular tissues, and found to effectively gather information on a significant portion of equine genes.
Objectives and Methods
- The aim of the research was to develop a unique GeneChip microarray that can explore the equine transcriptome and decipher the genetic underpinnings of equine diseases.
- Researchers utilized equine mRNA and certain equine gene sequences that were identified through successful cross-hybridisation of equine RNA on human microarray GeneChips for the design of the custom microarray.
- A glass-slide based gene chip was created using this sequence data, which covered multiple exons from the 5′-3′ spectrum.
- To study the performance of this custom microarray, it was characterised with RNA derived from various organs, as well as from cultured cells of chondrocytes, cartilage, synovial tissue, and stem cells.
Results
- The equine specific gene microarray was able to probe more than 12,300 equine genes simultaneously.
- When checked with RNA from parenchymatous organs and articular tissues, successful results were obtained 61.7% and 62.8% of the time, respectively.
- It was found to provide expression information on up to 90% of crucial molecules involved in signalling, metabolism, and developmental pathways.
- Unexpectedly high levels of certain types of collagen were found in normal articular cartilage, reflecting expression from the epiphyseal layers of maturing articular epiphyseal cartilage.
Conclusions
- A unique oligonucleotide microarray with over 12,300 probe sets was successfully generated.
- This was achieved by combining a labelling strategy that used expressed sequence tags from the entire transcriptome and supplementing selected human sequences that had the ability to cross-hybridise with horse genes.
- The microarray displayed robust performance upon testing and thus may prove valuable in understanding the onset and progression of equine diseases.
Cite This Article
APA
Gläser KE, Sun Q, Wells MT, Nixon AJ.
(2009).
Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage.
Equine Vet J, 41(7), 663-670.
https://doi.org/10.2746/042516409x412381 Publication
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
MeSH Terms
- Animals
- Cartilage, Articular / metabolism
- Gene Expression Profiling / veterinary
- Gene Expression Regulation
- Growth Plate / metabolism
- Horses / genetics
- Horses / metabolism
- Microarray Analysis / veterinary
- Oligonucleotide Array Sequence Analysis / veterinary
Citations
This article has been cited 2 times.- Bourgeois MA, Denslow ND, Seino KS, Barber DS, Long MT. Gene expression analysis in the thalamus and cerebrum of horses experimentally infected with West Nile virus. PLoS One 2011;6(10):e24371.
- Brosnahan MM, Brooks SA, Antczak DF. Equine clinical genomics: A clinician's primer. Equine Vet J 2010 Oct;42(7):658-70.
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