Tissue (liver, spleen, gills) samples were fixed in RNAlater (cat.no R0901, Sigma-Aldrich, Denmark), placed at 4°C for 24 h and subsequently stored at -20°C until further processing.
Growth protocol
Fish were maintain in plastic tanks with low density. Water was recycled (20 L/min) by internal biofilters (EHEIM, Germany) applying 30% water replenishment daily. Water quality was monitored and kept constant at pH 7.6, nitrite < 0.01 mg/l, nitrate < 50 mg/l (TetraTest 6in1, cat.no. T704154, Eldorado A/S, Denmark) and ammonia < 0.5 mg/l (API Ammonia Test Strips, VioVet Ltd, UK). During the 14 days acclimation and 14 days experimental infection period, fish received commercial pelleted feed (1.5% biomass per day, INICIO 917, BioMar A/S, Denmark). After the pathogen exposure fish with clinical signs were euthanized with overdose (300 mg/L) of ethyl-3-aminobenzoate methanesulfonate (MS222) (cat.no. A5040, Sigma-Aldrich, Denmark)
Extracted molecule
total RNA
Extraction protocol
RNAlater fixed samples from fish (gills, liver and spleen) were homogenized (Tissue-lyser II, Qiagen, Denmark) using a homogenization buffer with 2-mercaptoethanol (Sigma-Aldrich). RNA was extracted using the GenEluteTM mammalian RNA kit (RTN350, Sigma-Aldrich, Denmark). Liver samples were pre-treated by Proteinase K (cat.no.P4850, Sigma-Aldrich, Denmark). DNase I (AMPD1, Sigma-Aldrich, Denmark) treatment was applied to remove genomic DNA. The concentration of RNA was determined in a NanoDrop 2000 spectrophotometer (Saveen & Werner, Denmark). Quality of RNA was evaluated by agarose (cat.no. 16500100, ThermoFisher Scientific, Denmark) gel electrophoresis. RNA was kept at −80°C until cDNA synthesis.
Label
FAM
Label protocol
cDNA synthesis was performed in T100 thermocycler, Biorad, Denmark, using Oligo d(T)16 primer and TaqMan® Reverse Transcription Reagents (cat.no. N8080234, Thermo Fischer Scientific, Denmark). Finally, cDNA was stored at −20°C until further use. Quantitative PCR assays were performed using an AriaMx Real-Time PCR machine (cat.no.G8830A-04R-010, AH diagnostics AS, Denmark). The cycling conditions were one cycle of predenaturation at 95°C for 3 min. This was followed by 40 cycles of denaturation at 94°C for 5 sec with a combined annealing/elongation process at 60°C for 15 sec with endpoint measurement. Primers and Taq-Man probes targeting immune-relevant rainbow trout genes were synthesized at TAG Copenhagen AS, Denmark. Reaction volumes were 12.5 μl (2.5 μl cDNA, 6.25 μl Brilliant III Ultra-Fast QPCR Master Mix (600881, AH Diagnostics AS, Denmark), 1.0 μl primer-probe mixture (forward primer, 10 μM and reverse primer, 10 μM), Taq-Man probe (5 μM), and 2.75 μl RNase-free water. Reverse transcriptase minus and negative controls were used for every plate setup. . NormFinder (Andersen et al., 2004) was applied to evaluate all combinations of three reference genes (Elongation factor (ELF) 1-α, β-actin and acidic ribosomal phosphoprotein P0 (ARP)) as endogenous control; the average of the three genes was chosen and stability values were 0.002, 0.005 and 0.002 for liver, spleen and gills, respectively.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Replica 1
Data processing
All q-PCR assays exhibited efficiencies within 100% ± 5% so the simplified 2-ΔΔCq method was used for analysing data (Livak and Schmittgen, 2001). For normalization and as internal calibrator the average of three genes (ARP, ELF1α and β-actin) was chosen by using NormFinder (Andersen et al., 2004). For gene expression analyses, all challenged groups were compared to non-exposed time point controls using Student’s t-test. We considered differences to be significant when regulations were at least two-fold and p < 0.05. For four of the genes investigated in the non-exposed timepoint control groups less than three fish showed Cq values (excluding a t-test) and therefore we applied a qualitative assessment (presence/absence of Cq values) and analyzed data with the nonparametric Mann-Whitney test using a probability level of 5%.
Real-time quantitative PCR analysis of rainbow trout immune related gene expressed during exposure to Vibrio anguillarum
Data table header descriptions
ID_REF
VALUE
Target gene signals were normalized using the average of 3 housekeeping genes (ARP, b-actin and ELF 1a) as2^-ΔCt, where -ΔCt = -(Ct_Target − Ct_ average of 3 HKGs). Folds were calculated as 2^-ΔΔCt, where -ΔΔCt = -[ΔCtinfected -ΔCtUninfected] using idividual uninfected time point controls. No fold changes was calculated for samples timepoint zero since there were no infected group at that timepoint.
