Total RNA from RNAlater preserved salmon head-kidney samples was extracted using TRI reagent (Molecular Research Center, OH, USA) followed by column purification using the NucleoSpin RNA II (Macherey-Nagel, PA, USA) and following the manufacturer’s protocol. An on column DNase digestion step was performed on each sample. RNA quantification was performed using a NanoDrop 8000 (Thermo Scientific, DE, USA) and RNA quality and integrity was assessed with the Experion automated electrophoresis system (Bio-Rad, TX, USA). Only RNA samples with RNA Quality Indicator (RQI) values above 8 were used for analysis. RNA’s were stored at -70ºC in DEPC treated water containing an RNase inhibitor (Qiagen, ON, Canada) until needed.
Label
Cy3
Label protocol
RNA amplification and labeling for microarray was performed using the one-color Low Input Quick Amp Labeling kit (Agilent, CA, USA) and following the manufacturer’s recommendations. Briefly, 200 ng of each RNA sample was reverse transcribed, amplified and labeled using Cy3 dyes resulting in Cy3 labeled amplified RNA (aRNA). Purification of the labeled aRNA was performed using NucleoSpin RNA clean up columns (Macherey-Nagel, CA, USA) following the manufacturer’s recommendations. Following purification, labeled aRNA samples were then quantified using the NanoDrop 8000 to verify the yield of aRNA and the labeling efficiency. 1.65 µg of each purified aRNA was prepared for hybridization using the fragmentation step described in Agilent’s Low Input Quick Amp labeling kit protocol. Briefly each aRNA was fragmented by mixing 1.65 µg of aRNA in a volume of 22.8 µl, with 6 µl of 10X blocking agent and 1.2 µl of 25X fragmentation buffer and heating the mix at 60°C for 30 min.
Hybridization protocol
Microarray hybridizations were performed using Agilent’s 4 x 44K Atlantic salmon microarray slides containing 4 grids with the same ~ 44 000 features (Design ID 020938), using a one-color approach. All hybridizations, including washes and drying steps were carried out on a HS4800 automated hybridization station using quad chambers (Tecan, Männedorf, Switzerland). Briefly, slides were initially washed with Agilent’s aOligo aCGH Prehybridization buffer for 1 min at 65°C and injected with a 60 µl of hybridization mix containing 30 µl of fragmentation mix and 30 µl of Agilent’s 2x GEx hybridization Buffer Hi-RPM per chamber. The injected material was hybridized for 17 hour at 65°C. Following hybridization slides were washed twice with Agilent’s gene expression wash buffer 1 at 23°C for 1 min, twice with Agilent’s gene expression wash buffer 2 with 0.01 wash buffer additive at 37°C for 1 min and then dried under nitrogen
Scan protocol
Microarray slides were scanned at a resolution of 5 µm using a ScanArray Gx microarray scanner (Perkin Elmer, MA, USA) with the laser power set at 90%. For each slide, PMT settings were set to 60.
Description
252093810051_1_r10-97
Data processing
TIFF images were quantified using the SpotReader feature extraction software (Niles Scientific, CA, USA). Numerical intensities from each spot were extracted and descriptive statistics were calculated and exported in a GenePix Results (GPR) file. Features that did not meet certain default criteria in SpotReader were flagged as bad. These default criteria included flagging spots which had more than 50 % of the pixels saturated, spots with less than 8 pixels in diameter and spots with high background The bad flag values computed were then used to filter out genes that had missing values (-50,-75,-100 GPR flag values) on more than half of the slides used in each group (e.g. H5Rc fish) for a particular statistical analysis performed. Median intensity value minus background for each spot remaining after filtering were log2 transformed and normalized using a median centering approach.
