Investigation Title	Transcription profiling of mouse embryos 1.5, 3 and 6 hr after maternal treatment with the teratogen sodium valproate	
Comment[Submitted Name]	Transcription profiling of valproic acid induced gene expression in mice embryos	
Experimental Design	compound_treatment_design	co-expression_design	in_vivo_design	transcription profiling by array	
Experimental Design Term Source REF	mo	mo		EFO	
Comment[ArrayExpressReleaseDate]	2008-03-28	
Comment[AEMIAMESCORE]	5	
Comment[ArrayExpressAccession]	E-MEXP-1062	
Comment[MAGETAB TimeStamp_Version]	2010-08-09 19:38:57 Last Changed Rev: 13058	
Experimental Factor Name	dose	time	compound	
Experimental Factor Type	dose	time	compound_treatment_design	
Experimental Factor Term Source REF	
Person Last Name	Jergil	
Person First Name	Måns	
Person Mid Initials	
Person Email	Mans.Jergil@farmbio.uu.se	
Person Phone	 46184714252	
Person Fax	 46184714253	
Person Address	not available	
Person Affiliation	Pharmaceutical Biosciences	
Person Roles	submitter	
Person Roles Term Source REF	The MGED Ontology	
Quality Control Type	biological_replicate	dye_swap_quality_control	
Quality Control Term Source REF	The MGED Ontology	The MGED Ontology	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2008-03-28	
PubMed ID	
Publication DOI	
Publication Author List	
Publication Title	
Publication Status	
Publication Status Term Source REF	
Experiment Description	The antiepileptic drug valproic acid (vpa) is known teratogen giving neural tube defects (ntd:s). Administration of vpa to female NMRI on gestation day 8 induces a high incidence of ntd. In this study we investigate the time dependent gene expression changes induced in the embryo 1.5, 3 respectively 6 hr after maternal sodium valproate treatment.	
Protocol Name	P-MEXP-50543	P-MEXP-50544	P-MEXP-50545	P-MEXP-50546	P-MEXP-50548	P-MEXP-50547	P-MEXP-50549	P-MEXP-50562	P-MEXP-50566	
Protocol Type	grow	specified_biomaterial_action	pool	nucleic_acid_extraction	labeling	labeling	hybridization	feature_extraction	bioassay_data_transformation	
Protocol Description	Mice of the strain NMRI were kept on a 12-h light cycle (11 AM-11 PM) in the Laboratory Animal Facility at the Biomedical Center, Uppsala, Sweden. Females were mated with males for 2 h at the end of the dark period (8 AM-10 AM). Females were checked for vaginal plugs, and the midpoint of the mating period was taken as time of conception.	On day 8.0 past conception (8.0 dpc), pregnant dams were subjected to intraperitoneal injection of 600 mg/kg body weight sodium valproate (Sigma) in 0.9% saline; control mice received saline only. For microarray analysis, embryos were harvested in PBS (pH 7.4), 1.5, 3 respectively 6 h post treatment (8.25 dpc) and then transferred to Trizol reagent (Invitrogen), homogenized and stored at -70°C until further use.	Pooling_embryos To get sufficient amount of total RNA and overcome variations between litters, total RNA origin from embryos from three dams were pooled for each treatment and time. Pool 1_1.5 hr_control: embryos from mice 35+43+47. Pool 2_1.5 hr_control: 39+40+48. Pool 3_1.5 hr_treated: 36+37+46. Pool 4_1.5 hr_treated: 41+42+45. Pool 5_3 hr_control: 60+61+65. Pool 6_3 hr control: 59+62+63. Pool 7_3 hr_treated: 50+56+57. Pool 8_3 hr_treated: 49+51+55. Pool 9_6 hr_control: 22+23+28. Pool 10_6 hr_control: 32+33+34. Pool 11_6 hr_treated: 24+26+27. Pool 12_6 hr_treated: 25+29+30. 	Freeze stored Trizol lysates were thawed and separated by addition of chloroform and subsequent centrifugation. The upper aqueous phase were then mixed with isopropyl alcohol and centrifuged at 12000 g for 10 min to precipitate RNA. Total RNA was washed once with 75% ethanol and solved in RNase-free water, concentration determined with NanoDrop and quality checked with Agilent bioanalyzer, and subsequently stored in -70 °C.	Briefly, 0.5 µg total RNA was mixed with T7 Promotor Primer and with cDNA master mix (first strand buffer, DTT, dNTP mix, MMLV RT and RNaseOUT) and incubated at 40˚C for 2 hrs to synthesis cDNA. cDNA was then mixed with either cyanine 3-CTP or cyanine 5-CTP and subsequently mixed with Transcription Master Mix (Nuclease-free water, Transcription buffer, DTT, nTP mix, 50 % PEG, RNaseOUT, inorganic Pyrophosphatase and T7 RNA Polymerase) and incubated at 40˚C for 2 hrs. cRNA was recovered using Qiagen´s RNeasy Mini Kit (Qiagen, Hilden, Germany) and concentration determination was made with NanoDrop instrument.	Briefly, 0.5 µg total RNA was mixed with T7 Promotor Primer and with cDNA master mix (first strand buffer, DTT, dNTP mix, MMLV RT and RNaseOUT) and incubated at 40˚C for 2 hrs to synthesis cDNA. cDNA was then mixed with either cyanine 3-CTP or cyanine 5-CTP and subsequently mixed with Transcription Master Mix (Nuclease-free water, Transcription buffer, DTT, nTP mix, 50 % PEG, RNaseOUT, inorganic Pyrophosphatase and T7 RNA Polymerase) and incubated at 40˚C for 2 hrs. cRNA was recovered using Qiagen´s RNeasy Mini Kit (Qiagen, Hilden, Germany) and concentration determination was made with NanoDrop instrument.	0.75 µg cyanine 3-labelled cRNA and 0.75 µg cyanine 5-labelled cRNA (control and treated or vice versa) were mixed with Control Targets, nuclease-free water and Fragmentation Buffer and incubated at 60˚C for 30 minutes to fragment the cRNA. Fragmented cRNA was then mixed with Hybridization Buffer and hybridized on Agilent oligo microarray at 60˚C for 17 hrs. After washes with Wash solution (nuclease-free water, SSC and Triton X-102) arrays were dried using nitrogen-filled air gun.	Scanning of Agilent oligo microarrays was carried out on a GenePix 4000B scanner (Axon Instruments) with a pixel size of 10µm using GenePix Pro 5.1.0.4, utilizing the option to find irregular features. Scan power was set to 100% and the photomultiplier tube (PMT) voltage settings were adjusted to completely saturate less than 0.1% of the spots and give about the same total intensity in both channels. 	To remove systematic sources of variation for the Agilent microarrays, global loess normalization was done (Yang et al., 2002), in which different types of quality control spots were removed and the relative weight of 0.1 was given to spots flagged as missing or bad. Global loess normalization was used instead of local loess normalization, since all spots are spotted in a single block. Although all four arrays for each time point studied compare a treatment with a control, the arrays are not independent. To take account the fact that RNA from each pool of embryos appears on two different arrays a linear model (Smyth, 2004) was fitted with a coefficient for each sample and then a contrast was extracted from treated and control samples for each individual time point. To remove replicate spots with high variation, spots with a standard deviation >1.3 for each time point was also removed. The normalized data are presented as log2-ratios.	
Protocol Parameters				Amplification;Extracted product;	Amount of nucleic acid labeled;Label used;Amplification;	Amplification;Label used;Amount of nucleic acid labeled;	time;Quantity of label target used;Chamber type;temperature;Volume;	
Protocol Hardware								GenePix 4000B [Axon Instruments]	
Protocol Software								GenePix Pro [Axon Instruments]	
Protocol Contact	
Protocol Term Source REF		mo	The MGED Ontology		mo	mo		The MGED Ontology	The MGED Ontology	
SDRF File	E-MEXP-1062.sdrf.txt	
Term Source Name	The MGED Ontology		mo	ncbitax	ArrayExpress	The MGED Ontology	mo	EFO	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version