Development and validation of an oligonucleotide microarray for immuno-inflammatory genes of ruminants.
This report describes the development of small DNA microarrays of fully defined genes suitable for projects requiring detailed analysis of gene expression in sheep and/or cattle. Two arrays have been developed; the first is a small reference microarray (RIGRA) that has been used to validate experimental design and methodology; the second, a larger array (RIGUA) containing probes for 516 ruminant immuno-inflammatory genes, each represented by non-overlapping 75mer oligonucleotides. Experiments used to validate this microarray were: (1) a comparison of gene expression profiles from sheep broncho-alveolar macrophages before and after in vitro activation with lipopolysaccharide (LPS), using the RIGRA; (2) the differential gene expression between five in vitro unstimulated sheep keratinocyte cultures; (3) LPS/interferon gamma stimulated and unstimulated blood monocytes purified from Holstein-Friesians (Bos taurus) and Sahiwals (Bos indicus) cattle using the RIGUA. Real-time, quantitative RT-PCR was used to validate the gene expression profiles obtained with the RIGUA microarrays. The potential for using such an immunological tool in understanding the relative gene expression corresponding to immune-inflammatory responses of sheep and cattle is discussed.
Moredun Research Institute, Pentland Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZ, UK.
Craig
C
Watkins
Annie
A
McKellar
Kirsty
K
Jensen
Abraham
A
George
Doug
D
Jones
Michael J
MJ
Sharp
Karen
K
Stevenson
John
J
Hopkins
D000818:Animals; D002417:Cattle / Q000235:genetics* / Q000276:immunology*; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods / Q000662:veterinary*; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000662:veterinary; D015603:Keratinocytes / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000235:genetics; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012756:Sheep / Q000235:genetics* / Q000276:immunology*
D008070:Lipopolysaccharides