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Sample GSM146830 Query DataSets for GSM146830
Status Public on Nov 22, 2007
Title Blue catfish Liver ESC-infection 3d replicate3
Sample type RNA
 
Source name Blue catfish Liver 3d post ESC infection
Organism Ictalurus furcatus
Characteristics Livers from ~15 cm long D&B strain blue catfish
Biomaterial provider Auburn University, Department of 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.
 
Submission date Nov 22, 2006
Last update date Nov 22, 2006
Contact name Eric Peatman
E-mail(s) peatmer@auburn.edu
Organization name Auburn University
Department Fisheries and Allied Aquacultures
Lab Fish Molecular Genetics and Biotechnology Laboratory
Street address 203 Swingle Hall
City Auburn
State/province AL
ZIP/Postal code 36849
Country USA
 
Platform ID GPL4476
Series (1)
GSE6350 Transcriptomic profiling of the livers of blue catfish (Ictalurus furcatus) after infection with Edwardsiella ictaluri

Data table header descriptions
ID_REF
VALUE Average normalized signal of at least 6 perfect match oligos
SE_EXPRS Standard error of the mean of the PM observations

Data table
ID_REF VALUE SE_EXPRS
CATF0001S00000001 155.1132 0.8074
CATF0001S00000002 184.1072 0.846
CATF0001S00000003 179.0798 0.8222
CATF0001S00000004 567.5659 0.9393
CATF0001S00000005 4823.2777 0.961
CATF0001S00000006 43.3845 0.7767
CATF0001S00000007 428.546 0.8642
CATF0001S00000008 670.4936 0.884
CATF0001S00000009 421.353 0.9204
CATF0001S00000010 849.746 0.871
CATF0001S00000011 644.9172 0.9335
CATF0001S00000012 302.3626 0.8558
CATF0001S00000013 335.9304 0.7985
CATF0001S00000014 182.9658 0.8301
CATF0001S00000015 127.3586 0.8467
CATF0001S00000016 271.1773 0.8239
CATF0001S00000017 341.6212 0.8389
CATF0001S00000018 1965.5124 0.9564
CATF0001S00000019 89.9536 0.8162
CATF0001S00000020 410.0479 0.9434

Total number of rows: 28504

Table truncated, full table size 934 Kbytes.




Supplementary file Size Download File type/resource
GSM146830.ftr.gz 7.8 Mb (ftp)(http) FTR

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