Quantitative real-time PCR (RT-qPCR) is more sensitive, specific, rapid and accurate than traditional semi-quantitative and northern hybridization methods. It can more accurately analyse differences in gene expression and has become the most commonly used method for the determination of gene expression level. Up to now, few studies have focused on the stability of reference genes in Tetraodontidae. The purpose of this study was to use geNorm, BestKeeper and Normfinder software to select appropriate reference genes in the brain, liver, spleen, kidney, muscle and gill tissues of Takifugu rubripes under normal conditions and conditions that stimulated by Cryptocaryon irritans. The research shows that the differences among algorithms may produce inconsistent results. The results of various algorithms showed that the most stable reference gene in normal head kidney and liver wasRPL7, the most stable reference genes in normal spleen and gill were EF1A and B2M, and the optimal reference genes in normal brain and muscle were ACTB and GAPDH respectively. In the infected group, the most stable reference genes for muscle and gill were EF1A and ACTB, respectively, while the most stable reference genes for kidney and liver were RPL7 and B2M. Our verification experiment also proved this. This study provides a basis for identifying the functions of immune-related genes at the transcriptional level and, at the same time, helps to further analyse the pathogenic mechanism of expression of T. rubripes stimulated by C. irritans. The results also provide appropriate reference genes for the study of T. rubripes.

中文翻译：

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在正常条件下和白点虫感染后通过定量 RT-qPCR 分析 Takifugu rubripes 对潜在参考基因的评估

定量实时 PCR (RT-qPCR) 比传统的半定量和 Northern 杂交方法更灵敏、特异、快速和准确。它可以更准确地分析基因表达的差异，已成为测定基因表达水平最常用的方法。到目前为止，很少有研究集中在Tetraodontidae中参考基因的稳定性。本研究的目的是利用geNorm、BestKeeper和Normfinder软件，在正常条件下和刺激白点虫刺激条件下，选择合适的参考基因，分别在Takifugu rubripes的脑、肝、脾、肾、肌肉和鳃组织中进行。. 研究表明，算法之间的差异可能会产生不一致的结果。各种算法的结果表明，正常头部肾脏和肝脏中最稳定的参考基因是RPL7，正常脾脏和鳃中最稳定的参考基因是EF1A和B2M，正常脑和肌肉中最佳的参考基因是ACTB和GAPDH。分别。在感染组中，肌肉和鳃最稳定的参考基因分别是EF1A和ACTB，而肾脏和肝脏最稳定的参考基因是RPL7和B2M. 我们的验证实验也证明了这一点。本研究为在转录水平上鉴定免疫相关基因的功能提供了基础，同时有助于进一步分析T表达的致病机制。 由C刺激的rubripes。 刺激物。该结果也为T的研究提供了合适的参考基因。 红宝石。