Immune Gene Expression in Tongue Sole

Tongue sole as described above were randomly divided into three groups and administered via intraperitoneal injection with 50 µl PBS containing 6.25 µM NKLP27 or P86P15. The control group was injected with 50 µl PBS. At 1 h, 12 h, and 24 h post-injection, tissues were collected and used for total RNA extraction with the RNAprep Tissue Kit (Tiangen, Beijing, China). One microgram of RNA was used for cDNA synthesis with the Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA). The expression levels of interleukin (IL)-1β, IL-8, CsCCK1, CsCXCe1, toll-like receptor 9 (TLR9), myeloid differentiation factor 88 (Myd88), CsISG15, and CsG3BP were determined by qRT-PCR with primers reported previously [9] (link), [38] (link)–[40] (link). qRT-PCR was carried out in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously [41] (link). Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The expression levels of the immune genes were analyzed using comparative threshold cycle method with beta-actin as the control.