Investigation Title	Transcription profiling of male flounder isolated from different rivers
Comment[Submitted Name]	Flounder Environmental Tyne vs Alde Male
Experimental Design	growth_condition_design	transcription profiling by array
Experimental Design Term Source REF	mo	EFO
Comment[ArrayExpressReleaseDate]
Comment[AEMIAMESCORE]	3
Comment[ArrayExpressAccession]	E-MEXP-14
Comment[MAGETAB TimeStamp_Version]	2010-08-09 21:36:44 Last Changed Rev: 13058
Experimental Factor Name	sampling_site
Experimental Factor Type	sampling_site
Experimental Factor Term Source REF
Person Last Name	Minchin	Gensberg	Chipman	Williams	Williams
Person First Name	Steven	Karl	James	Timothy	Tim
Person Mid Initials	D		K	D	D
Person Email					t.d.williams@bham.ac.uk
Person Phone					0121 414 3393
Person Fax					0121 414 5925
Person Address					Edgbaston,Birmingham,West Midlands,B15 2TT,United Kingdom
Person Affiliation	Biosciences,The University of Birmingham	Biosciences,The University of Birmingham	Biosciences,The University of Birmingham	Biosciences,The University of Birmingham	Biosciences,The University of Birmingham
Person Roles					submitter
Person Roles Term Source REF					mo
Quality Control Type	biological_replicate
Quality Control Term Source REF
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date	2005-01-01
PubMed ID	12946615
Publication DOI	12946615
Publication Author List	Williams TD, Gensberg K, Minchin SD, Chipman JK
Publication Title	A DNA expression array to detect toxic stress response in European flounder (Platichthys flesus)
Publication Status	journal_article
Publication Status Term Source REF	mo
Experiment Description	Comparison of environmentally sampled flounder from the Tyne and Alde estuaries, related experiment E-MEXP-15
Protocol Name	P-MEXP-1028	P-MEXP-1027	P-MEXP-1029	P-MEXP-1030	P-MEXP-1031	P-MEXP-1035
Protocol Type
Protocol Description	Flounder were sampled from the polluted Tyne estuary (Tyne and Wear, UK) and the clean reference site, the Alde estuary (Suffolk, UK). Caught fish were placed in tanks containing flowing estuarine water, those over 15cm in length were killed by cervical dislocation. Livers were removed and immediately frozen in liquid nitrogen. Samples were subsequently stored at -80C. 	Feral European flounder (Platichthys flesus) were sampled from UK estuaries. Fish were caught during November 2000 by staff from CEFAS (Centre for Environment, Fisheries and Aquaculture Science).	Flounder liver samples were crushed with DEPC treated, autoclaved, mortars and pestles. mRNA was prepared from each sample with the PolyAttract kit (Promega). First strand cDNA was synthesised from 200ng mRNA using Superscript II reverse transcriptase (Invitrogen) with random nonamer primers (Alta Bioscience, Birmingham, UK). Remaining RNA was removed by adding NaOH (Sigma) to 133 mM and EDTA (Sigma)to 33mM and incubating at 65C for 1 hour. Subsequently, the reactions were neutralised with Tris/Cl pH8 (Sigma) added to 250mM. cDNA was purified by QIA-prep spin columns (Qiagen), after addition of sodium acetate pH 5.2 (Sigma) to 333mM.  cDNA was eluted in 50ul EB (Qiagen).	cDNA was mixed with 20ul 2.5x Random Priming mix (from Bioprime kit, Invitrogen), made up to 41ul with sterile water. This was boiled 5 min, put on ice. 5ul 10x dNTPs (1.2mM each dNTP except dCTP at 0.6mM), 1ul Cy3-dCTP (AP Biosciences)and 1ul Klenow polymerase (40 U) (from Bioprime kit, Invitrogen)were added and incubated at 37C for 2-18 hours.  To purify the probe, 5ul 3M sodium acetate pH 5.2 was added then Qiagen Qia-prep PCR cleanup spin columns were used according to the manufacturer’s protocol. Samples were eluted with 100ul or 200ul EB (Qiagen). Absorbance of each reaction was checked at 550nm versus EB blank. 	cDNA was mixed with 20ul 2.5x Random Priming mix (from Bioprime kit, Invitrogen), made up to 41ul with sterile water. This was boiled 5 min, put on ice. 5ul 10x dNTPs (1.2mM each dNTP except dCTP at 0.6mM), 1ul Cy5-dCTP (AP Biosciences)and 1ul Klenow polymerase (40 U) (from Bioprime kit, Invitrogen)were added and incubated at 37C for 2-18 hours. To purify the probe, 5ul 3M sodium acetate pH 5.2 was added then Qiagen Qia-prep PCR cleanup spin columns were used according to the manufacturer’s protocol. Samples were eluted with 100ul or 200ul EB (Qiagen). Absorbance of each reaction was checked at 650nm versus EB blank	Mixed probes (40 pmoles of cDNA-incorporated Cy3 and Cy5 as measured by absorbance) were concentrated using a Microcon YM30 filter (Amicon)then made up to 10ul with sterile distilled water.  Prehybridisation solution (25% formamide, 5x SSC, 0.1% SDS, 10mg/ml BSA (fraction V), filtered) was preheated to 42C in a Coplin jar. Slides were incubated at 42C for 2hr. Slides were dunked briefly in water, then in ethanol, placed in 50ml Falcon tubes, centrifuged  in a swing-out rotor at 2000 rpm 10min at room temperature to dry, then used immediately for hybridization.  10ul of 2x hybridisation buffer (50% Formamide, 10x SSC, 0.2% SDS, 1.6 ug/ul Poly-A, 4 ug/ul yeast tRNA) was added to 10ul probe mix. Hybridization mix was heated to 95C for 3min, quickly centrifuged 30 sec, then pipetted onto array. A plastic coverslip (Sigma Hybrislip) was used. The slide was placed in a Corning hybridization chamber and hybridized for 16 hours at 42C in the dark. Slide was washed 5 min in 2xSSC/0.1% SDS at 42C (after coverslip removed), washed at room temperature in 0.1x SSC/0.1% SDS for 5 min, washed four times at room temp. in 0.1x SSC for 5 min each, quickly diped in two water washes and one ethanol wash then dried by centrifugation as above.
Protocol Parameters		min temperature;max temperature;media;stop Time;start Time;	Amplification;Extracted product;	Label used;Amount of nucleic acid labeled;Amplification;	Amplification;Label used;Amount of nucleic acid labeled;	Temperature;Volume;Time;Quantity of label target used;Chamber type;
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF
SDRF File	E-MEXP-14.sdrf.txt
Term Source Name	atcc		ArrayExpress	mo	EFO
Term Source File	http://www.atcc.org/		http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/
Term Source Version