Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.)
Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.)
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DOI：
10.1016/j.molimm.2005.07.009
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发表时间：
2006-03-01
影响因子：
3.6
通讯作者：
Wergeland, HI
中科院分区：
文献类型：
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作者：
Ingerslev, HC;Pettersen, EF;Wergeland, HI
在未受刺激的大西洋鲑鱼（Salmo salar L.）的7种不同组织和白细胞中，检测了三种常用的内务基因ef1 - α、RPS20和β - actin的表达水平。实时荧光定量PCR分析的组织为鳃、肝、肠、肌、脾、头肾白细胞（HKL）和外周血白细胞（PBL）。实验是为了研究组织和白细胞之间以及个体之间的转录稳定性。除肌肉组织外，所有组织和白细胞均鉴定出合适的内参基因。使用HKL作为校准器，在研究的6个组织和白细胞中，有5个组织和白细胞中ef1 - α的表达相对于18S rRNA的表达变化最大，达到2.5倍。RPS20基因更为中间，变异幅度至少为2倍，最大可达20倍。β -肌动蛋白通常是最受调节的基因，相对于校准器，在鳃（5.8倍）和脾组织（10.3倍）中表现出很高的变异。没有找到适合肌肉组织的内参基因，因为这三种基因的表达与校准物相比变化在8.3到25倍之间。通过Normfinder程序比较非刺激组织和白细胞的表达结果，进一步表明ef1 - α是个体和不同组织/白细胞之间表达最稳定的基因。此外，用脂多糖（LPS）刺激大西洋鲑鱼的TO细胞和HKL，以揭示免疫刺激剂是否会改变ef1 - α、RPS20和β - actin的表达水平。LPS对细胞的刺激表明，相对于18S rRNA， LPS对TO细胞中RPS20和ef1 - α的调节作用最小，而在HKL中，β - actin是最合适的基因。然而，在lps刺激的TO细胞和HKL中，变异总体上最大为两倍，这使得这三个基因都适合作为本病例的内参基因。进一步的实验表明，未受刺激的组织和细胞中不存在RT-和/或PCR抑制剂，表明真正的转录差异。(c) 2005 Elsevier Ltd版权所有。
The expression levels of three commonly used housekeeping genes, EF1-alpha, RPS20 and Beta-Actin, were examined in seven different tissues and leucocytes from non-stimulated Atlantic salmon (Salmo salar L.). The tissues analysed by quantitative real-time PCR were gill, liver, intestine, muscle, spleen, head kidney leucocytes (HKL) and peripheral blood leucocytes (PBL). The experiments were performed to investigate the transcriptional stability within and between tissues and leucocytes and between individuals. For all tissues and leucocytes, an appropriate reference gene was identified except for muscle tissue. HKL were used as a calibrator and the expression of EF1-alpha varied maximally 2.5-fold in five out of the six tissues and leucocytes investigated relative to the expression of 18S rRNA. The RPS20 gene was more intermediate and varied at least by a factor of two and maximally by a 20-fold factor. Beta-Actin was generally the most regulated gene showing high variations for gill (5.8 x) and spleen tissue (10.3 x) relative to the calibrator. A suitable reference gene for muscle tissue was not found since the expression varied between 8.3- and 25-fold for the three genes compared to the calibrator. By comparing the expression results of the non-stimulated tissues and leucocytes using the Normfinder programme, it was further shown that EF1-alpha was the most stably expressed gene both between individuals and the different tissues/leucocytes. Stimulation with lipopolysaccharide (LPS) of TO cells and HKL from Atlantic salmon was additionally performed to reveal whether an immune stimulating agent would change the expression level of EF1-alpha, RPS20 and Beta-Actin. LPS stimulation of cells revealed that RPS20 and EF1-alpha were least regulated by the LPS treatment in the TO cells relative to 18S rRNA, but in HKL, Beta-Actin was the most appropriate gene. However, the variations were overall maximally two-fold in LPS-stimulated TO cells and HKL, which make all three genes suitable as reference genes in this case. A further experiment showed that no RT- and/or PCR inhibitors were present in the non-stimulated tissues and cells, indicating true transcriptional differences. (c) 2005 Elsevier Ltd. All rights reserved.