Meanwhile, the combination of the reference genes Cq value and the overall final ranking with RefFinder suggested that EF1A and ACTB were the two best reference genes used together to normalize the expression level of IgM during vaccination and infection in spleen of tilapia, which was well in line with the reports that two reference genes are required for accurate normalization in spleen of tilapia with S. agalactiae and PBS infection [15]. This evidence concerns the gene ACTB and infection.