Investigation Title	Transcription profiling of wild type and Ahr(-/-) mouse embryo fibroblast cells	
Comment[Submitted Name]	Tomlinson-Ahr-KO MEF	
Experimental Design	dye_swap_design	genetic_modification_design	transcription profiling by array	
Experimental Design Term Source REF	mo	mo	EFO	
Comment[ArrayExpressReleaseDate]	2006-10-17	
Comment[AEMIAMESCORE]	3	
Comment[ArrayExpressAccession]	E-MEXP-787	
Comment[MAGETAB TimeStamp_Version]	2010-08-11 15:57:15 Last Changed Rev: 13058	
Experimental Factor Name	genotype	
Experimental Factor Type	genotype	
Experimental Factor Term Source REF	
Person Last Name	Wesselkamper	Leikauf	Sivaganesan	Medvedovic	Sartor	Tomlinson	
Person First Name	Scott	George	Siva	Mario	Maureen	Craig	
Person Mid Initials	C	D			A	R	
Person Email					sartorma@umich.edu	
Person Phone					513-558-6781	
Person Fax	
Person Address					3223 Eden Ave	
Person Affiliation	University of Cincinnati	University of Cincinnati	University of Cincinnati	University of Cincinnati and CCHMC	Environmental Health	Dartmouth College	
Person Roles					submitter	
Person Roles Term Source REF						
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	2006-10-17	
PubMed ID	17177995	
Publication DOI	17177995	
Publication Author List	Maureen Sartor; Craig Tomlinson; Scott Wesselkamper; Siva Sivaganesan; George Leikauf; Mario Medvedovic	
Publication Title	Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarrays	
Publication Status	journal_article	
Publication Status Term Source REF	
Experiment Description	Wildtype vs. Ahr(-/-) mouse embryo fibroblast cells were compared in a flipped-dye design	
Protocol Name	P-MEXP-28923	P-MEXP-28924	P-MEXP-9172	P-MEXP-1378	P-MEXP-1379	P-MEXP-9164	P-MEXP-1381	
Protocol Type	grow	grow	nucleic_acid_extraction	labeling	labeling	hybridization	image_acquisition	
Protocol Description	Wildtype MEF cells were grown in culture at 37C in alpha-MEM media supplemented with 10% FBS and 1% penicillin strep for 1 to 4 days.	Ahr(-/-) MEF cells were grown in culture at 37C with alpha-MEM media supplemented with 10%FBS and 1% penicillin strep for 1-2 wks.	Total RNA was isolated using Tri Reagent (Invitrogen) according to the manufacturer's instructions with additional purification steps applied to RNA samples used for microarray analysis. To verify RNA quality, samples were analyzed using an Agilent 2100 Bioanalyzer.	Extracted total RNA concentration was 1-2 ug/ul with a volume of 10 ul. Oligo (dT) (IDT Technologies, Coralville, IA) was added at concentration of 2 ug/ul and volume of 2.5 ul and total solution volume was brought to 18 ul with nuclease-free water. Solution was denatured at 70°C for 10 min then placed on ice for 5 min. A 50X dNTP Stock Solution was made as follows: 10 ul each 100 mM dATP, dGTP, dCTP (Amersham, Piscataway, NJ); 8 ul 100 mM aminoallyl-dUTP (Sigma, St. Louis, MO) was made by dissolving 10 mg aminoallyl-dUTP in 170 ul nuclease-free water, adding approximately 6.8 ul 1N NaOH, and getting final pH of approximately 7.0 using pH paper; 2 ul 100 mM dTTP (Amersham, Piscataway, NJ). RT master mix was made according to Gibco Superscript II RT specifications for 500 U Superscript II RT (Gibco, Carlsbad, CA). In addition, 0.6 ul 50X dNTP Stock Solution and 0.5 ul of 40 U/ul RNAsin (Promega, Madison, WI) were added to the RT master mix. Once the sample cooled, 11.6 ul of RT master mix was added to the RNA sample reaction. The reaction was incubated for 1 hour at 42ºC. An additional 1 ul of Superscript II was added and the incubation continued at 42ºC for a further hour. RNA was degraded by addition of 15 ul of 0.1 N NaOH and incubation at 70°C for 10 min.  The solution was brought to room temperature and 15 ul of 0.1 N HCl was added to neutralize the reaction.  Then, 6.0 ul 3 M Sodium Acetate pH 5.2 and 150 ul cold absolute EtOH were added and it was stored at a minimum of -20°C overnight. Solution was then spun down at 13 K RPM at 4°C for 15 min. The supernatant was poured off and the pellet was washed with 750 ul cold 80% EtOH and spun again at 13 K RPM for 10 min. This wash was repeated and then the pellet was dried.  The pellet was then resuspended in 5 ul water and it was heated at 42°C for 10 min, vortexing and spinning every 5 min. Then, 5 ul of coupling buffer (0.1 M sodium bicarbonate, pH 9.0) was added to the sample. A tube of the monofunctional NHS-ester Cy3 (Amersham, Piscataway, NJ) was resuspended in 10 ul of 100% DMSO, brought to room temperature, and 1 ul of this dye was added to the sample. The reaction was incubated at room temperature in the dark for 1 hr, vortexing and spinning every 15 min. The reaction was quenched with 4.5 ul of 4 M hydroxylamine and incubated for an additional 15 min at room temperature in the dark. The paired samples, each labeled with their respective dye were then combined and 70 ul water and 6 ul of 3 M sodium acetate, pH 5.2, were added. The combined sample was cleaned up using the Qia-Quick PCR purification kit (Qiagen, Valencia, CA) by adding 500 ul Buffer PB, applying to the column supplied, and spinning at 13 K for 1 min. The flow-through was reapplied and the column was spun again. The flow-through was decanted and 750 ul of Buffer PE + Ethanol was added, spun through, decanted, and the column was spun dry for 1 additional min. The column was transferred to a 1.5 ml microfuge tube and 30 ul of Buffer EB was added and the column was incubated at room temperature in the dark for 5 min followed by another 1 min spin. This step was repeated with 30 ul water and the total 60 ul flow-through was vacuum-dried for approximately 1 hr or until completely dry.	Extracted total RNA was at a concentration of 1-2 ug/ul with a volume of 10 ul. Oligo (dT) (IDT Technologies, Coralville, IA) was added at a concentration of 2 ug/ul and a volume of 2.5 ul and the total solution volume was brought to 18 ul with nuclease-free water. The solution was denatured at 70°C for 10 min then placed on ice for 5 min. A 50X dNTP Stock Solution was made as followed: 10 ul each 100 mM dATP, dGTP, dCTP (Amersham, Piscataway, NJ); 8 ul 100 mM aminoallyl-dUTP (Sigma, St. Louis, MO) was made by dissolving 10 mg aminoallyl-dUTP in 170 ul nuclease-free water, adding approximately 6.8 ul 1N NaOH, and getting final pH of approximately 7.0 using pH paper; 2 ul 100 mM dTTP (Amersham, Piscataway, NJ). The RT master mix was made according to the Gibco Superscript II RT specifications for 500 U Superscript II RT (Gibco, Carlsbad, CA). In addition, 0.6 ul 50X dNTP Stock Solution and 0.5 ul of 40 U/ul RNAsin (Promega, Madison, WI) were added to the RT master mix. Once the sample had cooled, 11.6 ul of the RT master mix was added to the RNA sample reaction. The reaction was incubated for 1 hr at 42ºC. An additional 1 ul of Superscript II was added and the incubation continued at 42ºC for an additional 1 hr. The RNA was degraded by addition of 15 ul of 0.1 N NaOH and incubation at 70°C for 10 min. The solution was brought to room temperature and 15 ul of 0.1 N HCl was added to neutralize the reaction. Then, 6.0 ul 3 M Sodium Acetate pH 5.2 and 150 ul cold absolute EtOH were added and it was stored at a minimum of -20°C overnight. The solution was then spun down at 13 K RPM at 4°C for 15 min. The supernatant was poured off and the pellet was washed with 750 ul cold 80% EtOH and spun again at 13 K RPM for 10 min. This wash was repeated and then the pellet was dried. The pellet was then resuspended in 5 ul water and it was heated at 42°C for 10 min, vortexing and spinning every 5 minutes. Then, 5 ul of coupling buffer (0.1 M sodium bicarbonate, pH 9.0) was added to the sample. A tube of the monofunctional NHS-ester Cy5 (Amersham, Piscataway, NJ) was resuspended in 10 ul of 100% DMSO, brought to room temperature, and 1 ul of this dye was added to the sample. The reaction was incubated at room temperature in the dark for 1 hr, vortexing and spinning every 15 minutes. The reaction was quenched with 4.5 ul of 4 M hydroxylamine and incubated for an additional 15 minutes at room temperature in the dark.  The paired samples, each labeled with their respective dye were then combined and 70 ul water and 6 ul of 3 M sodium acetate, pH 5.2, were added. The combined sample was cleaned up using the Qia-Quick PCR purification kit (Qiagen, Valencia, CA) by adding 500 ul Buffer PB, applying to the column supplied, and spinning at 13 K for 1 min. The flow-through was reapplied and the column was spun again. The flow-through was decanted and 750 ul of Buffer PE + Ethanol was added, spun through, decanted, and the column was spun dry for an additional minute. The column was transferred to a 1.5 ml microfuge tube and 30 ul of Buffer EB was added and the column was incubated at room temperature in the dark for 5 min followed by another 1 minute spin. This step was repeated with 30 ul water and the total 60 ul flow-through was vacuum-dried for approximately 1 hr or until completely dry.	Prehybridization buffer: 5x SSC, 0.1% SDS and 1% BSA (Sigma catalog# B-4287). Slides to be analyzed are placed in a staining jar, prehybridization buffer is added, and incubation is carried out at 48ºC for 45-60 min. The slides are washed and excess water is removed. The slides are then dipped 10 times in isopropanol and spun dried. A 2x hybridization mixture is prepared, containing 50% formamide, 10x SSC and 0.2% SDS at 48°C. Labeled targets are resuspended in 9 ul water and the mixture is heated to 95°C for 3 min to denature, and centrifuged at maximum angular velocity for 1 min. The following are added to each tube in order to block non-specific hybridization: COT1-DNA (1 mg/ml), 8 ul (Roche Diagnostics; Cat# 1581074), poly(A)-DNA (10 mg/ml), 2 ml (Sigma; Cat# P 9403), yeast tRNA (4 mg/ml), and 2 ul(Sigma; Cat# R 8759). The 21 ul 2x hybridization buffer is added to the target mixture, triturated well and centrifuged. The labeled target is applied to a prehybridized microarray slide and covered with a 22 x 60 mm glass cover slip. The slide is placed in a sealed hybridization chamber (Corning), and 12 ul water is added. The sealed chamber is placed in a 48°C water bath and incubated for 40-60 hrs. The slide is then placed in a rack for a staining dish containing 1x SSC and 0.2% SDS at 48°C. The coverslip is gently removed while the slide is in solution and agitated for 4 min. The slides are transferred to a staining dish containing 0.1x SSC and 0.2% SDS at room temperature and agitated for 4 min. The slides are transferred to a staining dish containing 0.1x SSC at room-temperature and agitated for 4 min twice. Slides are spun dried.	The GenePix 4000 Microarray Scanner (Axon Instruments, Union City, CA) was turned on, software was started, and the slide to be scanned was inserted into the scanner. PMTs are set to 500 in both the 635nm (Cy5) and the 532nm (Cy3) channels. A low resolution Preview Scan was performed in order to determine the location of spots to set the Scan Area around and the initial fluorescence intensities. A visual inspection of the fluorescence intensities within the adjusted Scan Area allows for initial adjustments to PMTs. Since these are dual-labeled, global gene-expression hybridizations, the ratio for the whole scan area was preferably near 1.0. Before actual data scan is performed, the scanner is set so that it scanned each pixel twice and averaged these data counts. This reduces background noise that may be present. A high-resolution scan is performed. During the scan, PMTs were fine-tuned, not just to achieve a normalized balance of 1.0 but also to have pixels represented across a broad dynamic range. Once PMT levels were set, a final Data Scan was performed.	
Protocol Parameters	start time;min temperature;max temperature;stop time;	start time;stop time;min temperature;max temperature;	Amplification;Extracted product;	Amplification;Label used;Amount of nucleic acid labeled;	Amplification;Amount of nucleic acid labeled;Label used;	Quantity of label target used;time;Volume;temperature;Chamber type;	
Protocol Hardware							Axon GenePix 4000B scanning hardware	
Protocol Software							GenePix	
Protocol Contact	
Protocol Term Source REF	
SDRF File	E-MEXP-787.sdrf.txt	
Term Source Name	NCI_thesaurus		mo	ncbitax	The MGED Ontology	ArrayExpress	mo	EFO	
Term Source File	ncithesaurus.obo.alt		http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	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/	
Term Source Version