Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage
Version of Record online: 5 JAN 2010
2009 EVJ Ltd
Equine Veterinary Journal
Volume 41, Issue 7, pages 663–670, September 2009
How to Cite
GLÄSER, K. E., SUN, Q., WELLS, M. T. and NIXON, A. J. (2009), Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage. Equine Veterinary Journal, 41: 663–670. doi: 10.2746/042516409X412381
- Issue online: 5 JAN 2010
- Version of Record online: 5 JAN 2010
- [Paper received for publication 28.05.08; Accepted 13.12.08]
- gene expression;
- whole transcript labelling assay;
- mRNA hybridisation;
- cartilage gene expression
Reasons for performing study: 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.
Objectives: 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′ multiexon-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.
Conclusion: 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.
Potential relevance: This array may be a useful tool to elucidate the pathogenesis of equine diseases.