Real-time quantitative PCR analysis of rainbow trout immune related gene expressed during exposure to Vibrio anguillarum

Total RNA was extracted from V. angularium susceptible and resistant rainbow trout, tissues (liver, spleen, gill), in different time points using GenEluteTM mammalian RNA kit (RTN350, Sigma-Aldrich, Denmark). After measuring quantity (NanoDrop 2000 spectrophotometer (Saveen & Werner, Denmark)) and quality (gel electrophoresis) of RNA, cDNA was synthetised in T100 thermocycler, Biorad, Denmark, using Oligo d(T)16 primer and TaqMan® Reverse Transcription Reagents (cat.no. N8080234, Thermo Fischer Scientific, Denmark). Primers and probes for total of 28 genes including three housekeeping genes were synthesized at TAG Copenhagen AS, Denmark. qPCR reactions were run by Brilliant III Ultra-Fast QPCR Master Mix (600881, AH Diagnostics AS, Denmark) for all samples. The fold changes analysed by the simplified 2-ΔΔCq method. Fingerlings of rainbow trout (mean body weight of 12 g) were exposed (2 h bathing, 18°C) to the pathogen V. anguillarum serotype O1 in a solution of 1.5x107 cfu/ml and observed for 14 d. Disease signs appeared three days post exposure (dpe) whereafter morbidity progressed exponentially until 6 dpe reaching a total morbidity/mortality of 55% within 11 days. we sampled fish for immune gene expression analysis when they first showed clinical signs, fish without clinical signs at the same time point and finally fish surviving the exposure to the pathogen. The different immune gene expression profiles in the different groups were addressed when discussing possible resistance mechanisms in rainbow trout. 15 rainbow trout with clinical signs (CS), 15 rainbow trout with no clinical signs (NCS) at the same time point (3 days post exposure), 15 rainbow trout surviving the challenge (11 days post exposure) were samples and for each time point and time point zero (before the challenge started) 15 rainbow trout were sampled from uninfected fish as control group for normalizing the data. Organs liver, spleen and gills were samples from all groups.