Selection of stable reference genes for RT–qPCR in Salmo trutta

To identify suitable reference genes for Salmo trutta, real-time fluorescent quantitative PCR (RT–qPCR) technology was used to determine the expression of eight candidate genes in 11 tissues (heart, liver, spleen, head kidney, muscle, brain, gills, ovary, intestine, skin, testis) of S. trutta. Concurrently, expression of these eight reference genes was assessed in 1 d and 30 d S. trutta embryos and muscle tissue of 90 d and 180 d S. trutta. Finally, the eight candidate genes were comprehensively screened using four types of screening software (GeNorm, NormFinder, BestKeeper and RefFinder), and the stability of their expression in different tissues, in response to bacterial stress and during different developmental stages in S. trutta tissues was ranked. The results indicated that the eight candidate genes were specifically amplified in each tissue of S. trutta, with band sizes ranging from 73 bp to 150 bp. The stability ranking of the eight candidate genes in different tissues of healthy adult S. trutta was hprt1>β-actin>EF1a>RPL13a>RPS29>β2M>GAPDH>28 S rRNA. The ranking of the stability of expression in different tissues of S. trutta artificially infected with Aeromonas salmonicida was RPS29>hprt1>β2M>RPL13a>EF1a>β-actin>28 S rRNA>GAPDH. The ranking of the stability of expression in the tissues of S. trutta at different developmental stages was RPS29>GAPDH>hprt1>RPL13a>EF1a>28 S rRNA>β-actin>β2M. Comprehensive analysis revealed that expression of the hprt1 gene was relatively stable under different conditions. These results could guide the quantitative analysis of functional gene expression in S. trutta and calibrate the expression of major functional genes of S. trutta, facilitating the acquisition of more accurate, reliable and meaningful results.