individual: fish 3 cell type: Macrophage-like cells isolated from fish head kidney developmental stage: Pre-adult agent: PBS
Treatment protocol
[cell isolation and stimulation] Each fish was netted and immediately euthanized with an over-dose of MS222 (400 mg/L; Syndel Laboratories, Vancouver, BC). Each fish was then dissected, and the head kidney was removed and minced through 100 µm nylon cell strainers. The cell suspension (in L-15) was centrifuged on a discontinuous 25/51% Percoll gradient (GE Healthcare, Uppsala, Sweden) at 300 × g for 40 min at 4 ˚C, and the macrophage-enriched interface was collected. The resulting macrophage-rich suspension was washed twice in L-15 by centrifuging at 300 × g for 15 min at 4 ˚C. Thereafter, the cells seeded into 35 mm culture plates. The cells were allowed to adhere overnight (16 h) at 10˚C, and then the non-adherent cells were removed by washing the culture plates 3 times with L-15. The isolated macrophages from each individual were incorporated in all experimental groups. Macrophages were exposed to either 50 µg/ml pIC or PBS (control) at 24 h post-isolation. The stimulated and non-stimulated macrophages were harvested using TRIzol at 24 h post-stimulation
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from TRIzol-lysed samples, following the manufacturer’s instructions, and RNA pellets were dissolved in 50 µl Dnase/Rnase-free water. The RNA samples were treated with Dnase-I (Qiagen, Mississauga, Ontario, Canada) for removing any residual genomic DNA contamination, and then purified using RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer’s instructions. The high-purity RNA samples (e.g. A260/230 and A260/280 ratios > 1.8) with tight 18S and 28S ribosomal RNA bands were used for the microarray analysis.
Label
Cy5
Label protocol
Macrophages exposed to 50 µg/ml pIC or PBS (control) for 24 h (pIC and PBS treatments for each of 6 individuals) were used for microarray analysis, using a common reference design and the Atlantic cod 20K oligonucleotide microarray platform. The mRNA of each sample was amplified and then labelled using 1 µg of DNase-treated and column-purified RNA and Amino Allyl MessageAmp™ II aRNA Amplification kit (Ambion®, Carlsbad, CA, USA), according to the kit’s procedure. A common reference was prepared using a pool of all 12 samples (15 µg aRNA from each sample). The experimental samples (pIC and control) were labelled with Cy5 (GE Healthcare, Buckinghamshire, UK), whereas the common reference samples were labelled with Cy3 (GE Healthcare), based on the manufacturer’s protocol.
Channel 2
Source name
pool of RNAs from all 12 samples involved in the microarray experiment
cell type: Macrophage-like cells isolated from fish head kidney developmental stage: Juvenile sample type: common reference
Treatment protocol
[cell isolation and stimulation] Each fish was netted and immediately euthanized with an over-dose of MS222 (400 mg/L; Syndel Laboratories, Vancouver, BC). Each fish was then dissected, and the head kidney was removed and minced through 100 µm nylon cell strainers. The cell suspension (in L-15) was centrifuged on a discontinuous 25/51% Percoll gradient (GE Healthcare, Uppsala, Sweden) at 300 × g for 40 min at 4 ˚C, and the macrophage-enriched interface was collected. The resulting macrophage-rich suspension was washed twice in L-15 by centrifuging at 300 × g for 15 min at 4 ˚C. Thereafter, the cells seeded into 35 mm culture plates. The cells were allowed to adhere overnight (16 h) at 10˚C, and then the non-adherent cells were removed by washing the culture plates 3 times with L-15. The isolated macrophages from each individual were incorporated in all experimental groups. Macrophages were exposed to either 50 µg/ml pIC or PBS (control) at 24 h post-isolation. The stimulated and non-stimulated macrophages were harvested using TRIzol at 24 h post-stimulation
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from TRIzol-lysed samples, following the manufacturer’s instructions, and RNA pellets were dissolved in 50 µl Dnase/Rnase-free water. The RNA samples were treated with Dnase-I (Qiagen, Mississauga, Ontario, Canada) for removing any residual genomic DNA contamination, and then purified using RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer’s instructions. The high-purity RNA samples (e.g. A260/230 and A260/280 ratios > 1.8) with tight 18S and 28S ribosomal RNA bands were used for the microarray analysis.
Label
Cy3
Label protocol
Macrophages exposed to 50 µg/ml pIC or PBS (control) for 24 h (pIC and PBS treatments for each of 6 individuals) were used for microarray analysis, using a common reference design and the Atlantic cod 20K oligonucleotide microarray platform. The mRNA of each sample was amplified and then labelled using 1 µg of DNase-treated and column-purified RNA and Amino Allyl MessageAmp™ II aRNA Amplification kit (Ambion®, Carlsbad, CA, USA), according to the kit’s procedure. A common reference was prepared using a pool of all 12 samples (15 µg aRNA from each sample). The experimental samples (pIC and control) were labelled with Cy5 (GE Healthcare, Buckinghamshire, UK), whereas the common reference samples were labelled with Cy3 (GE Healthcare), based on the manufacturer’s protocol.
Hybridization protocol
For each array, 3 µg of labelled aRNA of one individual, 3 µg of labelled aRNA of the common reference and 2 µl of LNA blocker (Genisphere, Hatfield, Pennsylvania, USA) were mixed in a formamide-based hybridization buffer (Genisphere) and hybridized to the array overnight (~ 16 h) at 42˚C. The detailed protocols for hybridizations and washing were described in Booman et al. (2011, Mar Biotechnol, 13:733-50).
Scan protocol
Arrays were scanned at a resolution of 5 μm using a ScanArray Gx Plus scanner and ScanExpress v4.0 software (v4.0; Perkin Elmer, Woodbridge, ON) with photomultiplier tube (PMT) set to balance fluorescence signal between channels.
Data processing
Signal intensity data was extracted from tiff images using Imagene v9.0 (BioDiscovery). In addition to automated quality flagging, spots affected by dust particles, scratches and other local artefacts were flagged manually. Raw data was read into Bioconductor package 'marray', base 2 logratios were calculated from background-corrected median signals and control spots were removed. Data was normalized using printtip loess. Logratio data for a particular spot was removed when the raw signal value in one of the channels of that spot was lower than the threshold, which was calculated as (median background + 3 * SD) for each channel on each array. Logratio data for a particular spot was also removed if the spot was flagged in Imagene.
