Characterization of the expression stability of largemouth bass (Micropterus salmoides) candidate reference genes by qRT-PCR during viral infection

Quantitative Real-time PCR (qRT-PCR) is one of the most widely used methods for gene quantification because it is extremely sensitive, can be highly sequence specific, and has high precision while being easily reproducible in samples. The stability of the reference genes used for data normalization is crucial for exact experimental results and conclusions. In this study, qRT-PCR was used to analyze the expression of eight candidate reference genes including β-actin (ACTB), α-tubulin (TUBA), Elongation factor-1-α (EF1A), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), Hypoxanthine guanine phosphoribosyl transferase1 (HPRT), Ribosomal protein L7 (RPL7), and RNA polymerase II (RNA PoL Ⅱ) in largemouth bass. The expression and stability of above genes were evaluated among eight tissues with or without largemouth bass virus (LMBRaV) infection and assessed using BestKeeper, GeNorm, NormFinder statistical softwares and comparative Ct method, and then analyzed the comprehensive ranking by ReFinder. Our results showed that ACTB was identified as the most suitable genes of different tissues, while TUBA was ranked as the best reference gene in tissues infection with LMBRaV. To verify the screened reference genes, the expression pattern of IFN in spleen during LMBRaV infection was examined. This study provides useful information for gene expression characterization using qRT-PCR, which will benefit for future studies of gene regulation in largemouth bass.