Comment[ArrayExpressAccession]	E-GEOD-3261
Investigation Title	Global gene expression in catfish spleen after injection of lipopolysaccharide
Comment[Submitted Name]	Global gene expression in catfish spleen after injection of lipopolysaccharide
Publication Title	Production and utilization of a high-density oligonucleotide microarray in channel catfish, Ictalurus punctatus.
Publication Author List	Li RW, Waldbieser GC
Publication DOI	
PubMed ID	16740160
Experimental Design
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Public Release Date	2005-11-30
Experiment Description	A high-density oligonucleotide microarray for channel catfish (Ictalurus punctatus) was designed and produced with Maskless Array Synthesizer technology (MAS). The microarray contained ~379,652 24-mer oligonucleotides covering ~18,999 catfish unique sequences. The global expression profiling of the catfish spleens stimulated by lipopolysaccharide (LPS) for 2h, 4h, 8h and 24h was investigated with the microarray.  In the spleen samples, 409 genes were identified to be induced or repressed greater than 2-fold by LPS treatment.   Self-organizing maps (SOM) clustering analysis was applied to interpret gene expression patterns, and 84 of these genes were clustered into six expression patterns.  Real-time RT-PCR was used to verify the microarray results for 9 selected genes representing different expression levels. The results from real-time RT-PCR were positively correlated (R^2 = 0.87) with the results from the microarray. Channel catfish were injected with LPS or PBS carrier.  Fish were killed by anesthesia overdose at 2, 4, 8, or 24h post-injection and total RNA was isolated from spleen.  The experiment contained two replicate fish per time point.  Relative signal intensities for each feature were generated using the Robust Multi-Array Average algorithm and log2 transformed.  Transformed data were then processed using quantile normalization, and the average and std. error for each gene were calculated from the 10 perfectly matched oligos only.  After detransformation, the ratio (fold change) was calculated using PBS treated samples (control) as the baseline against LPS-treated samples.												
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Term Source Name	EFO
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Term Source File	http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl												
Person Last Name	Waldbieser	Li	Waldbieser										
Person First Name	Geoffrey	Robert	Geoff										
Person Mid Initials	C.												
Person Email	gwaldbieser@msa-stoneville.ars.usda.gov												
Person Affiliation	U.S. Dept of Agriculture												
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Person Address	Agricultural Research Service, U.S. Dept of Agriculture, 141 Experiment Station Road, Stoneville, MS, USA												
Person Roles	submitter												
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Protocol Name	P-GSE3261-1	P-GSE3261-6	P-GSE3261-13	P-GSE3261-9	P-GSE3261-3	P-GSE3261-11	P-GSE3261-10	P-GSE3261-8	P-GSE3261-7	P-GSE3261-2	P-GSE3261-12	P-GSE3261-4	P-GSE3261-5
Protocol Description	ID_REF = Gene identification number (Nimblegen)<br>VALUE = Average normalized signal of 10 perfectly matched oligos<br>HEADER_3 = Standard error of mean of 10 observations	Microarrays were pre-hybridized with 1X MES hybridization buffer (100mM MES, 1.0M Na+, 20mM EDTA, 0.01% Tween20) plus 40 µg of herring sperm DNA and 200 µg of acetylated BSA at 45°C for 15 min, followed by hybridization in the same buffer with 10µg of denatured and fragmented cRNA per microarray at 45°C for 16 – 20h with constant rotation. After hybridization, the microarrays were immediately washed extensively with non-stringent wash buffer (6x SSPE, 0.01% Tween20) at RT, and stringent wash buffer (100mM MES salt and free acid solution, 0.1M Na+, 0.01% Tween20) at 45°C. After final rinsing with non-stringent wash buffer, the microarrays were stained with 1x Stain buffer (100mM MES, 1M Na+, 0.05% Tween20, 50mg/ml of BSA, and 1mg/ml of Cy3-streptavidin) at room temp. for 25min. After the stain buffer was removed, the microarrays were rinsed with non-stringent wash buffer and immediately dried under argon gas.	Fish fed commercial diet to apparent satiation until 24h prior to injection.  Fish were killed by anesthesia overdose at 2, 4, 8, and 24h post-injection, spleen was dissected and transferred to Trizol reagent	Fish fed commercial diet to apparent satiation until 24h prior to LPS injection.  Fish were killed by anesthesia overdose at 4h post-injection, spleen was dissected and transferred to Trizol reagent	Fish fed commercial diet to apparent satiation until 24h prior to LPS injection.  Fish were killed by anesthesia overdose at 2h post-injection, spleen was dissected and transferred to Trizol reagent	Fish fed commercial diet to apparent satiation until 24h prior to LPS injection.  Fish were killed by anesthesia overdose at 24h post-injection, spleen was dissected and transferred to Trizol reagent	Fish fed commercial diet to apparent satiation until 24h prior to LPS injection.  Fish were killed by anesthesia overdose at 8h post-injection, spleen was dissected and transferred to Trizol reagent	The data was extracted from the raw images with NimbleScan software (Nimblegen, Inc.).  Relative signal intensities for each feature were generated using the Robust Multi-Array Average algorithm and log2 transformed.  Transformed data were then processed using quantile normalization, and the average and std. error for each gene were calculated from the 10 perfectly matched oligos.  After detransformation, the ratio (fold change) was calculated using PBS treated samples (control) as the baseline against LPS-treated samples.	The microarrays were scanned with an Axon GenePix 4000B scanner (Molecular Devices Corp., Union City, CA) at 5 µM resolution.	LPS from E. coli 0127:B8 (Sigma Aldrich) was resuspended in PBS (ph7.0).  Fish were anesthetized with tricaine methanesulfonate and injected intraperitoneally with LPS at 2.5 mg/kg body weight.	Fish were anesthetized with tricaine methanesulfonate and injected intraperitoneally with 0.5ml PBS.	Total RNA was purified according to Invitrogen protocol for Trizol reagent, treated with DNase1 and further purified using an RNease Mini kit (Qiagen).	The 1st strand cDNA was synthesized from 2.5ug total RNA using SuperScript II reverse transcriptase and 100 pmoles T7 promoter-oligo dT primer.  Complementary RNA was then synthesized with a MEGAscript in vitro Transcription kit (Ambion) containing biotin-16 UTP and biotin-11 CTP.  The biotinylated cRNA products were fragmented to 50-200 bp prior to hybridization.
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Protocol Type	bioassay_data_transformation	hybridization	grow	grow	grow	grow	grow	feature_extraction	image_aquisition	specified_biomaterial_action	specified_biomaterial_action	nucleic_acid_extraction	labeling
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Comment[SecondaryAccession]	GSE3261												
Comment[GEOLastUpdateDate]	2005-09-06
Comment[GEOReleaseDate]	2005-12-01
Comment[ArrayExpressSubmissionDate]	2005-09-06
SDRF File	E-GEOD-3261.sdrf.txt