Stability evaluation of reference genes for real-time PCR in zebrafish (Danio rerio) exposed to cadmium chloride and subsequently infected by bacteria Aeromonas hydrophila

• Cd exposure affects the stability of reference genes for real-time PCR in zebrafish.
• Reference genes present different stability in the five tissues (spleen, kidney, liver, gills and intestine) of zebrafish after Cd exposure.
• Bacterial infection further affects the stability of reference genes in Cd-treated zebrafish.

Environmental and occupational cadmium (Cd) toxicity is a global concern, and the model organism zebrafish is an ideal species to investigate Cd toxicity. Among various detecting techniques, quantitative real-time PCR (qPCR) is a sensitive and efficient tool. Stable reference genes are critical for relative qPCR analysis. However, accumulated evidence shows that conventional reference genes can vary significantly under different experimental setups. Here we evaluated the stability of eight candidate reference genes of zebrafish with or without exposure to different concentrations of Cd. The results showed that the best four suitable reference genes in the five selected organs were: (1) spleen: β-actin > gapdh > ef1α > rpl13α; (2) kidney: rplp2 > rpl7 > β-actin > ef1α; (3) liver: rpl7 > rpl13α > β-actin > ef1α; (4) gills: rplp2 > gapdh > rnf7 > ef1α; (5) intestine: ef1α > rnf7 > rplp2 > rpl13α. Moreover, we further assessed the expression stability of the four reference genes for Cd immunotoxicology studies in zebrafish. The expression profiles showed that ef1α in spleen and kidney, rpl13a in liver and rplp2 in intestine were the most suitable reference genes at 12 h and 9 days after the injection with Aeromonas hydrophila following Cd exposure. In gills, the expression of gapdh was more stable than ef1α after 9 days of bacteria challenge while ef1α showed a higher stability than gapdh at 12 h after bacteria injection. In conclusion, this study has demonstrated that different tissues of zebrafish have different suitable reference genes after Cd exposure and the subsequently pathogenic insults for qPCR. It emphasized the importance of reference gene evaluation for studies using qPCR, in particular when investigations involve factors not explored previously.