Institute of Marine Research, PO Box 1870 Nordnes, N-5817 Bergen, Norway.
BMC Mol Biol. 2010 May 11;11:36. doi: 10.1186/1471-2199-11-36.
Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, beta-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes.
The expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes.
Generally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.
实时 RT-PCR 已成为分析鱼类基因表达的重要工具。尽管已经在大西洋比目鱼（Hippoglossus hippoglossus L.）中评估了几种管家基因，但尚未确定在早期发育阶段用于低拷贝 mRNA 转录本的合适参考基因。尚未有报道尝试在感染 NNV 的比目鱼或刺激的比目鱼肝白细胞中鉴定合适的参考基因。在这项研究中，β-肌动蛋白 1（ACTB1）、延伸因子 1α（EF1A1）、次黄嘌呤-鸟嘌呤磷酸核糖基转移酶 1（HPRT1）、核糖体蛋白 L7（RPL7）、微管蛋白β 2C（Tubb2C）和泛素连接酶（UbcE）被评估为大西洋比目鱼发育过程中、健康鱼和 NNV 感染鱼的组织以及体内和体外刺激的前肾白细胞中实时 RT-PCR 数据归一化的参考基因。
在未受精卵至 12 天受精后（ddpf）期间，所有 6 个基因的表达都相对稳定。然而，在比目鱼发育的整个过程中，没有发现任何选定的基因表达稳定。6 个基因的 mRNA 水平从 18ddpf 开始增加，此时合子转录可能被激活，并在不同的时间点稳定下来。基于 Excel 的软件程序 BestKeeper、geNorm 和 NormFinder 将 EF1A1 和 UbcE 评为合子转录激活前最佳候选参考基因，RPL7 和 EF1A1 评为孵化后最佳候选基因。在研究各种比目鱼器官中 6 个基因的表达水平时，EF1A1 和 RPL7 也被列为最佳参考基因，无论是在未注射的鱼还是在模拟和 NNV 注射的鱼中。在体外刺激的前肾白细胞中，没有一种参考基因可用于归一化实时 RT-PCR 数据。
总的来说，发现 EF1A1 和 RPL7 是变化最小的基因，HPRT1 和 UbcE 是中间基因，ACTB1 和 Tubb2C 是最不稳定的基因。在 ConA-PMA 刺激的白细胞中，没有一种参考基因可以被推荐为参考基因候选物。然而，UbcE 可以成为其他实验设置的一个很好的候选物。这项研究强调了参考基因评估的必要性，因为尚未确定通用的参考基因。
