Selection of internal reference gene for quantitative real‐time PCR analysis of gene expression in largemouth bass ( Micropterus salmoides ) under different experimental conditions

Abstract

The study of gene function in largemouth bass requires accurate normalization by use of suitable internal reference genes. The expression levels of four candidate internal reference genes including β-actin, EF1-α, cytb and 12SrRNA in different tissues, two different developmental stages, and different experimental conditions of largemouth bass were evaluated by quantitative real-time PCR (qPCR) method. The expression stabilities of four candidate internal reference genes were analysed by geNorm, NormFinder and BestKeeper software calculation. The results showed that cytb is the best internal reference gene in different tissues and two different developmental stages of largemouth bass. The cytb and EF1-α was the best choice for internal reference gene in gill of largemouth bass 10 g and 55 g weighing under different salt concentrations respectively. After being challenged by Lipopolysaccharide (LPS), Aeromonas hydrophila and Edwardsiella tarda in largemouth bass weighing 55 g, EF1-α was recommended as the most suitable internal reference gene in spleen, but cytb was ranked as the best internal reference gene in head kidney. The expression levels of CaM normalized by EF1-α and cytb in spleen and head kidney of largemouth bass weighing 55 g, and which showed the significant upregulation in LPS group than those in the control group. Therefore, these results indicated that cytb and EF1-α are the suitable internal reference genes in different tissues, two different developmental stages and different experimental conditions respectively. This study will provide convincing information for the selection of suitable internal reference genes in different tissues, different developmental stages and different experimental conditions of largemouth bass.