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.

Validation and Functional Analysis of Reference and Tissue-Specific Genes in Adipose Tissue of Freshwater Drum, Aplodinotus grunniens, under Starvation and Hypothermia Stress

“…GAPDH plays a vital role in glycolytic processes [41] and has also been shown to be unsuitable as a common reference gene in adipose tissue due to its differential expression levels in brown adipose tissue (BAT) and white adipose tissue (WAT) [42]. In addition to the above-mentioned classical internal reference genes, many studies have also found that RPL7 [43,44] and Rpl13α [45,46] can also be used as internal references. In the present study, 60SRP was the most stably expressed gene in freshwater drum adipose tissue.…”

Identification of reference genes for quantitative expression analysis in Indian catfish,Clarias magur, under physiological and pathological conditions

“…Yang et al (2017) also reported 18S/ EF‐1α as stable RG in intestine of A. hydrophila infected group and 18S in PBS injected control group of Onychostoma Macrolepis . Liu et al (2021) reported EF‐1α and β‐actin as the most stable reference genes for muscle and gill in parasite infected tiger puffer fish. Information generated in this study can be used by research to study relative quantification of immune genes such as pathogen recognition receptor (PRR, i.e.…”