Investigation Title Transcription profiling of cod to determine whether gene expression profiles would reflect environmental levels and composition of pollutants in two saltwater recipients, Sarfjorden and Store Lungegardsvann Comment[Submitted Name] CodStress Experimental Design reference_design compound_treatment_design transcription profiling by array Experimental Design Term Source REF The MGED Ontology EFO Comment[ArrayExpressReleaseDate] 2009-06-01 Comment[AEMIAMESCORE] 3 Comment[ArrayExpressAccession] E-BASE-11 Comment[MAGETAB TimeStamp_Version] 2010-07-29 16:17:10 Last Changed Rev: 13058 Experimental Factor Name Location Pollution type Experimental Factor Type water external pollution of seawater Experimental Factor Term Source REF The MGED Ontology Person Last Name Lie Person First Name Kai Person Mid Initials K. Person Email kai.lie@nifes.no Person Phone Person Fax Person Address Person Affiliation National Institute of Nutrition and Seafood Research Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2009-06-01 PubMed ID Publication DOI Publication Author List Kai K. Lie?, Anders Lanzen?, Harald Breilid?, P?l A. Olsvik? Publication Title Gene expression profiling in Atlantic cod (Gadus morhua L.) from two contaminated sites using a custom made cDNA microarray Publication Status Publication Status Term Source REF Experiment Description In the present study we aimed to develop a custom made cDNA microarray, the CodStress array, for Atlantic cod to be used as a diagnostic tool in environmental monitoring of polluted waters. We wanted to investigate if the gene expression profiles would reflect environmental levels and composition of pollutants in two saltwater recipients, S?rfjorden and Store Lungeg?rdsvann. Protocol Name base.cbu.uib.no:PROT:Protocol-generic_sampling base.cbu.uib.no:PROT:Protocol-75 base.cbu.uib.no:PROT:Protocol-58 base.cbu.uib.no:PROT:Protocol-81 base.cbu.uib.no:PROT:Protocol-FS-3 Protocol Type unknown_protocol_type nucleic_acid_extraction labeling hybridization unknown_protocol_type Protocol Description This is a generic sampling protocol for experiments stored in the BASE system. For more details on the sampling process, see the action association of the treatment resulting in the sample of interest Total RNA was extracted in three steps 1. Modefied Phenol chloroform extraction (TRIZOL, Invitrogen, USA) were The upper aqueous phase was precipitated in 25% isopropanol and 25% RNA precipitation solution described by Chomczynski and Mackey 1995 (Biotechniques 19:942-945). 2. Genomic DNA was removed by DNase treatment using DNA-free (Ambion, Austin TX, USA). RNA was then precipitated over night in 75% ethanol and 2.5% 3M pH 5.2 NaAc. 3. An additionally purification step using RNeasy MiniElute Cleanup Kit (Qiagen, Hilden, Germany) was implemented The SuperScript? Indirect cDNA Labeling System is a highly efficient system for generating fluorescently labeled cDNA for use on microarrays in gene expression studies (De Risi et al., 1996; Eisen & Brown, 1999). It uses an aminoallyl-modified nucleotide and an aminohexyl-modified nucleotide together with other dNTPs in a cDNA synthesis reaction with SuperScript? III Reverse Transcriptase. After a purification step to remove unincorporated nucleotides, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye. A final purification step removes any unreacted dye, and the fluorescently labeled cDNA is ready for hybridization to microarrays. This system uses 5?20 ?g of total RNA or 0.4?2 ?g of mRNA as starting material, and is compatible with Alexa Fluor? 555 and Alexa Fluor? 647 fluorescent dyes from Invitrogen, Cy3? and Cy5? dyes from Amersham Biosciences, or other monoreactive NHS-ester dyes from a variety of manufacturers. The hybridization solution consists of 55 ?l of 2 x GE x Hybridization buffer HI-RPM (Agilent Technologies, Palo Alto, USA), 11 ?l of 10 x Blocking Agent (Agilent Technologies, Palo Alto, USA) and 44 ?l labeled cDNA. CodStress microarray slides are pre-hybridized at 65?C for one hour in 3.5 x SSC, 0.1% SDS and 10 mg/ml BSA in a 50 ml falcon tube. Samples/hybridization solutions are added to Hybridization Gasket Slides x 4 (cat G2534-6001 Agilent Technologies, Palo Alto, USA) Hybridization are performed at 60 ?C for 16 h. Following hybridization the slides were dipped in 2 x SSC and 0.1%SDS at 65 ?C to remove the gasket slide, then washed in 1 x SSC at 65 ?C for 5 min, 0.2 x SSC for 5 min. and 0.05 x SSC at room temperature for 45 seconds subsequently before drying by centrifugation. Generic protocol for Feature Extraction software Genepix. See SoftwareApplication for version, or MeasuredBioAssay/Data for other info Protocol Parameters Protocol Hardware Protocol Software Genepix Protocol Contact Protocol Term Source REF mo The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology SDRF File E-BASE-11.sdrf.txt Term Source Name The MGED Ontology ArrayExpress The MGED Ontology EFO mo Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version