antigen challenge: Phosphate-buffered saline (PBS) time point: 6 h post-injection diet treatment: High-w6
Treatment protocol
At the end of the feeding trial, salmon were lightly anesthetized (50 mg/L MS-222) and subsequently subjected to an intraperitoneal (IP) injection –at a dose of 1 μL/g of fish (wet mass)– with either phosphate-buffered saline (PBS; Ref# 20012027, Gibco/ThermoFisher Scientific, Mississauga, ON, Canada), a solution of polyriboinosinic polyribocytidylic acid [poly(I:C); Ref# P0913, Sigma-Aldrich, Oakville, ON, Canada], or a suspension of formalin-killed A. salmonicida (Furogen Dip, Novartis, Charlottetown, PE, Canada). The poly(I:C) solution was prepared in ice-cold and 0.2 μm-filtered PBS at 2 μg/μL. The ASAL suspension was prepared in ice-cold and 0.2 μm-filtered PBS, at an optical density of 1.0 at 600 nm wavelength. Head kidney samples were collected at 6 h or 24 h post-injection. At the sampling time points, salmon were euthanized by MS-222 overdose prior to head kidney collection. Head kidney samples were immediately flash-frozen with liquid nitrogen and stored at -80 ºC until processed for RNA extraction.
Growth protocol
Atlantic salmon post-smolts were fed either a high-soy oil diet (High-ω6) or a high-linseed oil diet (High-ω3) for 12 weeks. Fish holding conditions consisted of a flow-through filtered seawater system (12 L/min) and a 24-h light photoperiod; water quality was monitored daily and remained constant at 10.1 ± 0.2ºC [mean ± standard deviation (SD)] and 10.1 ± 0.5 mg O2/mL.
Extracted molecule
total RNA
Extraction protocol
The head kidney samples were homogenized in TRIzol using a motorized Kontes RNase-Free Pellet Pestle Grinder (Kimble Chase, Vineland, NJ, USA). Frozen homogenates were further processed by passing them through a QIAshredder (QIAGEN, Mississauga, ON, Canada) spin column following the manufacturer’s instructions. Total RNA was extracted using TRIzol (Invitrogen, Burlington, ON, Canada) following the manufacturer's instructions. Total RNA samples were treated with 6.8 Kunitz units of DNaseI (RNase-Free DNase Set, QIAGEN) with the manufacturer’s buffer (1X final concentration) at room temperature for 10 min. DNase-treated RNA samples were column-purified using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. RNA integrity was verified by 1% agarose gel electrophoresis, and RNA purity was assessed by A260/280 and A260/230 NanoDrop UV spectrophotometry. Column-purified RNA samples had A260/280 ratios between 2.0 and 2.2 and A260/230 ratios between 1.8 and 2.3.
Label
SYBR Green
Label protocol
First-strand cDNA templates for RT-qPCR were synthesized in 20 μL reactions from 1 μg of DNaseI-treated, column-purified total RNA using random primers (250 ng; Invitrogen), dNTPs (0.5 mM final concentration; Invitrogen), and M-MLV reverse transcriptase (200 U; Invitrogen) with the manufacturer’s first strand buffer (1X final concentration) and DTT (10 mM final concentration) at 37°C for 50 min. PCR amplifications were performed in 13 μL reaction volumes containing 1X Power SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies), 50 nM of both the forward and reverse primers, and the indicated cDNA quantity (see below). The PCR program consisted of 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min, with fluorescence detection at the end of each 60°C step and was followed by dissociation curve analysis. Primer amplification performance was tested using the 7500 Fast Real Time PCR system (Applied Biosystems). Single product amplification and absence of primer-dimer in the no-template control (NTC) were assessed via dissociation curve analysis. Amplicon size was checked electrophoretically on 2% agarose gels and compared with a 1 kb Plus DNA Ladder (Invitrogen). Amplification efficiencies were determined based on a 5-point 1:3 dilution series starting with cDNA representing 10 ng of input total RNA. RT-qPCR analyses of expression levels of 46 genes of interest (GOI) and 2 normalizer genes were performed using the ViiA 7 Real Time PCR system (384-well format) (Applied Biosystems). cDNA representing 4 ng of input total RNA was used as template in the PCR reactions. On each plate, for every sample, the GOIs and normalizer genes were tested in triplicate and a NTC was included. As expression levels of a given gene were measured across multiple 384-well plates, a plate linker sample (i.e., a sample that was run on all plates in a given study) was also included to ensure there was no plate-to-plate variability. The normalizer genes were selected from six candidate normalizers [60S ribosomal protein L32 (rpl32), β-actin (actb), elongation factor 1-alpha 1 (ef1a1), elongation factor 1-alpha 2 (ef1a2), eukaryotic translation initiation factor 3 subunit D (eif3d) and polyadenylate-binding protein cytoplasmic 1 (pabpc1)]. Briefly, the fluorescence threshold cycle (CT) values of 48 samples (i.e., 4 individuals for each diet and treatment at each time point) were RT-qPCR-measured in duplicate for each of these transcripts using cDNA representing 4 ng of input total RNA and the 7500 Fast Real Time PCR system (Applied Biosystems), and then analyzed using geNorm. Using this software, ef1a1 (geNorm M = 0.121) and rpl32 (geNorm M = 0.154) were determined to be stably expressed in all samples tested and thus selected as normalizers. The 384-well plate layout was designed to accommodate the simultaneous analysis of 3 genes of interest (GOIs) and the 2 normalizer genes. Each RT-qPCR experiment (i.e., each 3 GOI + 2 normalizer gene analysis) involved multiple linked plates . The completion of this research required a total of 16 independent RT-qPCR experiments. This analytical approach determined the structure of the data in the sheet Matrix non-normalized.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Control, 6 h post-injection, High-w6-fed fish
Data processing
The relative quantity (RQ) of each transcript was calculated using the ViiA 7 Software Relative Quantification Study Application (Version 1.2.3; Applied Biosystems). For each GOI, the RQ calculation considered the transcript levels of both normalizer genes for the normalisation, as well as the amplification efficiency, and set the lowest normalized expression value as the calibrator (i.e., assigned an RQ of 1.0).
