Watkins Craig, McKellar Annie, Jensen Kirsty, George Abraham, Jones Doug, Sharp Michael J, Stevenson Karen, Hopkins John
Moredun Research Institute, Pentland Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZ, UK.
Vet Res Commun. 2008 Dec;32(8):647-57. doi: 10.1007/s11259-008-9066-6. Epub 2008 Jun 20.
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.
本报告描述了适用于需要详细分析绵羊和/或牛基因表达项目的、由完全确定的基因组成的小型DNA微阵列的开发情况。现已开发出两种阵列；第一种是小型参考微阵列（RIGRA），已用于验证实验设计和方法；第二种是更大的阵列（RIGUA），包含516个反刍动物免疫炎症基因的探针，每个基因由不重叠的75聚体寡核苷酸代表。用于验证该微阵列的实验有：（1）使用RIGRA比较脂多糖（LPS）体外激活前后绵羊支气管肺泡巨噬细胞的基因表达谱；（2）五种体外未刺激的绵羊角质形成细胞培养物之间的差异基因表达；（3）使用RIGUA对从荷斯坦-弗里生牛（Bos taurus）和萨希瓦尔牛（Bos indicus）纯化的LPS/干扰素γ刺激和未刺激的血液单核细胞进行研究。采用实时定量RT-PCR来验证用RIGUA微阵列获得的基因表达谱。本文还讨论了使用这种免疫学工具来理解与绵羊和牛免疫炎症反应相对应的相对基因表达的潜力。
