Total RNA from RNAlater preserved salmon liver 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 025055), 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 Agilent’s High Resolution Microarray Scanner (Agilent, CA, USA) using a 20-bit data format and automatic PMT adjustment.
Data processing
The resulting TIFF images were quantified using Agilent’s Feature Extraction software (Agilent, CA, USA). Numerical intensities, descriptive statistics and flag value were computed for each spot and exported in a text file. Spots were considered flagged it they were saturated, not uniform, below background levels and if the background was not uniform. Genes that had a flag on more than 1/3 of the slides used for a particular statistical analysis were filtered out. The gProcessedSignal values (foreground value minus background value) for each spot were then log2 transformed and normalized between arrays using a median centering approach found in BRBArrayTool version 4.1.0 Beta_2 Release (developed by Dr. Richard Simon and BRB-ArrayTools Development Team). Statistical analysis was performed on log2 transformed and normalized gProcessedSignal value.
