Evaluation of potential reference genes by quantitative RT‐qPCR analysis of Takifugu rubripes under normal conditions and after Cryptocaryon irritans infection

Abstract

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.