Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage

Abstract

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' 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. 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.