RT-qPCR analysis of PoCD9.1 and PoCD9.3 expression during pathogen infections was performed as reported previously [41]. V. anguillarum and E. piscicida were cultured in LB broth at 28 °C to an optical density of 0.8 at 600 nm. Then, the cells were washed with phosphate-buffered saline (PBS) and resuspended in PBS to a concentration of 5 × 106 CFU (colony forming units)/mL. ISKNV was resuspended in PBS to a concentration of 1 × 106 copies/mL. The fish were divided randomly into four groups (16 fish per group) and injected intraperitoneally with 50 μL of V. anguillarum, E. piscicida, ISKNV, or PBS. After infection, the head kidney, spleen, and liver from three or four fish were taken aseptically at 6, 12, 24, 48, and 72 hours post-infection (hpi) for bacterial infection and at 1, 3, 5, and 7 days post-infection (dpi) for viral infection. PoCD9.1 and PoCD9.3 expression was determined by RT-qPCR, as described in the “Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis of PoCD9.1 and PoCD9.3 expression under normal conditions” section. To determine the stability of beta-actin as an internal reference, we detected the expression of beta-actin with EF1 alpha as an internal reference during bacterial and viral infections. The RT-qPCR results showed that beta-actin expression in the three examined tissues upon infection with different pathogens was not significantly changed (Additional file 1), indicating that beta-actin is stable under the current experimental conditions. The experiment was performed in triplicate.

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