National Diagnostics Centre, National University of Ireland, Galway, Ireland.
Comp Biochem Physiol Part D Genomics Proteomics. 2008 Mar;3(1):51-66. doi: 10.1016/j.cbd.2007.04.009. Epub 2007 May 18.
A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising "early" stress (2-48 h) and "late" stress (96-504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, beta-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.
利用专门设计的微阵列平台（Stressgenes，第 1 阶段），研究了虹鳟鱼在长期禁闭期间肝脏中基因表达的变化。在转移到标准化禁闭应激源后，每隔一段时间从鳟鱼中采集组织和血液样本，同时从未受干扰的对照鱼中采集匹配的样本。分析血浆中的 ACTH、皮质醇、葡萄糖和乳酸，以确认对禁闭的神经内分泌反应与先前的发现一致，并为解释基因表达数据提供表型背景。用于抑制性消减杂交（SSH）文库构建的肝样品选自包括“早期”应激（2-48 小时）和“晚期”应激（96-504 小时）的实验组中。为了减少四个 SSH 文库中的冗余并产生更高数量的独特克隆，进行了额外的减法。在打印阵列后，执行了一系列 55 次杂交，以覆盖 6 个时间点。在 2 小时、6 小时、24 小时、168 小时和 504 小时时，使用了 5 条单独的禁闭鱼和 5 条单独的对照鱼，仅在 0 小时时使用对照鱼。通过数据分析方法的组合生成了一份考虑整个时间过程中差异调节的 314 个克隆的初步清单，并且发现最显著的基因表达变化发生在 24 小时至 168 小时期间，到 504 小时时一般接近对照水平。在最初的 6 小时内，表达的变化很少。表达显著改变的基因列表主要由属于生物过程类别（对刺激的反应）和一个细胞成分类别（细胞外区）的基因组成，主要由所谓的急性期蛋白组成。在禁闭期间对肝组织中的基因表达谱进行分析，揭示了一些显著的聚类。主要模式包括在 24 小时及以后上调的基因，主要例子是触珠蛋白、β-纤维蛋白原和 EST10729。从六个 k-均值聚类中的每一个聚类中选择两个代表性基因进行 qPCR 验证。大多数经过测试的基因的微阵列和 qPCR 表达模式之间存在显著相关性。qPCR 分析显示，触珠蛋白在 24 小时时上调约 8 倍，在 168 小时时上调超过 13 倍。
