Liver from ~15 cm long Kansas Random strain channel catfish
Biomaterial provider
Auburn University Fisheries and Allied Aquacultures
Treatment protocol
Edwardsiella ictaluri bacteria were cultured from a single isolate (MS-S97-773) and used in a small test infection of several channel catfish. Bacteria were re-isolated from a single symptomatic fish and biochemically confirmed to be E. ictaluri, before being inoculated into brain heart infusion (BHI) medium and incubated in a shaker incubator at 28 °C overnight. The bacterial concentration was determined using colony forming unit (CFU) per ml by plating 10 μl of 10-fold serial dilutions onto BHI agar plates. At the time of challenge, the bacterial culture was added to the aquaria to a concentration of 4X108 CFU/ml. Water was turned off in the aquaria for 2 h of immersion exposure, and then continuous water flow-through resumed for the duration of the challenge.
Growth protocol
Fish were moved to challenge aquaria at least two weeks prior to challenge and 30 channel catfish and 30 blue catfish were added to each aquaria. Fish were fed a commercial diet to satiation prior to challenge and all fish were fed lightly during the course of the challenge. Continuous water flowthrough and supplemental aeration were provided. At 3 d fish were killed by anesthesia overdose and their livers dissected. The livers of 25 fish were pooled for each replicate, flash frozen in liquid nitrogen, and stored at -80°C until RNA extraction.
Extracted molecule
total RNA
Extraction protocol
The livers for each replicate were ground in liquid nitrogen by mortar and pestle to a fine powder and thoroughly mixed. Approximately 30 mg of tissue powder was homogenized in Buffer RLT Plus by passing the lysate several times through a 20-gauge needle fitted to a syringe according to the protocol of the RNeasy Plus Mini Kit (Qiagen). Following the manufacturer’s instructions, approximately 35 μg of total RNA was obtained from each extraction. RNA quality and concentration was checked by spectrophotometer analysis and gel electrophoresis. All extracted samples had an A260/280 ratio of greater than 1.8, and were diluted to 1 μg/μL.
Label
Biotin
Label protocol
Total RNA was converted to double-stranded cDNA using a SuperScript II cDNA synthesis kit (Invitrogen) and an oligo-dT primer containing the T7 RNA polymerase promoter. In vitro transcription (IVT) was carried out to produce biotin-labeled cRNA from cDNA using the MEGAscript T7 kit (Ambion). Briefly, 3 μL double-stranded cDNA was incubated with 7.5 mM ATP and GTP, 5.6 mM UTP and CTP, 1.875 mM bio-11-CTP and bio-16 UTP (Enzo) and 1x T7 enzyme mix in 1x reaction buffer for 16 h at 37˚C. The cRNA was then purified using an RNeasy mini kit (Qiagen, ). Before hybridization, cRNA was fragmented to an average size of 50 to 200 bp by incubation in a buffer of 100 mM potassium acetate, 30 mM magnesium acetate, and 40mM tris-acetate for 35 min at 94˚C. Fragmentation was measured using a Bioanalyzer 1000 (Agilent Technologies).
Hybridization protocol
The microarrays were prehybridized with a solution of 2x MES hybridization buffer (100 mM 2-morpholinoethanesulfonic acid, 1.0 M Na+, 20 mM EDTA, 0.01% Tween 20), 50 µg of herring sperm DNA, and 250 µg of acetylated bovine serum albumin (BSA) at 45°C for 15 min followed by hybridization with 10 µg of denatured and fragmented cRNA per microarray, 3.5 µl of CPK6 control oligo, 35 µg of herring sperm DNA, 175 µg of acetylated BSA, and 2X MES buffer at 45°C for 16–20 h with constant rotation. After hybridization, the microarrays were washed twice with nonstringent buffer (6x SSPE, 0.01% Tween 20) at room temperature followed by two stringent washes (100 mM MES salt and free acid solution, 0.1 M Na+, 0.01% Tween 20) at 45°C for 15 min each. After a final one minute rinse with nonstringent buffer, the arrays were placed into a 1x stain solution (100 mM MES, 1 M Na+, 0.05% Tween 20, 50 mg/ml BSA, and 1 µg/µl Cy3-streptavidin) at room temperature for 15 min, agitating every few minutes. The microarrays were removed from the stain solution and placed in fresh nonstringent wash buffer for one minute. They were then placed into Nimblegen’s proprietary final wash buffer for 30 s, and then immediately dried under a stream of argon gas.
Scan protocol
The microarrays were scanned with an Axon GenePix 4000B scanner (Molecular Devices Corp., Union City, CA) at 5 µM resolution.
Description
During the challenge, symptomatic treatment fish and control fish were collected and confirmed to be infected with E. ictaluri and pathogen-free, respectively, at the Fish Disease Diagnostic Laboratory, Auburn University.
Data processing
After extraction of data from raw images using the NimbleScan software (Nimblegen, Inc.), gene calls were generated using the Robust Multichip Average (RMA) algorithm (Irizarry et al. 2003) which takes into account only the PM oligos. RMA takes a background adjustment on the raw intensity scale, carries out quantile normalization (Bolstad et al. 2003), takes the log2 of the normalized background adjusted PM values, and then uses a linear model to estimate expression values on the log scale.
