ONTOLOGY SOURCE REFERENCE
Term Source Name	"CHEBI"	"BTO"	"EFO"	"NCBITAXON"	"XCO"	"ERO"	
Term Source File	"http://data.bioontology.org/ontologies/CHEBI"	"http://data.bioontology.org/ontologies/BTO"	"http://data.bioontology.org/ontologies/EFO"	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/XCO"	"http://data.bioontology.org/ontologies/ERO"	
Term Source Version	"78"	"20"	"111"	"2"	"33"	"13"	
Term Source Description	"Chemical Entities of Biological Interest Ontology"	"BRENDA Tissue and Enzyme Source Ontology"	"Experimental Factor Ontology"	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"Experimental Conditions Ontology"	"Eagle-I Research Resource Ontology"	
INVESTIGATION
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INVESTIGATION PUBLICATIONS
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INVESTIGATION CONTACTS
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STUDY
Study Identifier	"E-GEOD-8251"
Study Title	"Transcriptional profiling of rat liver after drug treatment [original title: Non-genotoxic Hepatocarcinogens]"
Study Description	"990 samples. 147 different compounds. [original description: There is no accurate and well-validated short-term test for non-genotoxic carcinogens, necessitating an expensive two year rodent bioassay before a risk assessment can begin. We have developed a short-term in vivo rat assay that predicts whether non-genotoxic chemicals are likely to induce hepatic tumors based on transcript profiles in the liver. Using a large independent test set, assay accuracy was found to be superior to existing pathological and genomic markers. Comparison of the test chemical's signature profile to reference carcinogens of known mechanism can also identify a potential mode(s) of action, allowing an early assessment of human cancer risk. Guidelines for commercial use: http://www.iconixbiosciences.com/guidelineCommUse.pdf Keywords: dose response, time course, compound treatment Treatment of male Sprague-Dawley rats with 147 non-genotoxic compounds at various doses and durations, in biological triplicate, along with vehicle-matched control animals. Liver samples were assayed for gene expression. Hepatocarcinogenic and non-carcinogenic compounds were included. A classifier for carcinogenicity was built on a training set of 25 carcinogens and 75 noncarcinogens (randomly selected compounds, maximum tolerated dose, 5 day timepoints), and tested on the remaining 47 compounds at various timepoints. A total of 990 samples were hybridized to single-channel CodeLink RU1 arrays. Biological triplicates were combined with matched control samples to calculate log ratios.]"
Comment[Study Grant Number]	""
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Study Submission Date	""
Study Public Release Date	"2007-06-22"
Study File Name	"s_E-GEOD-8251_study_samples.txt"
STUDY DESIGN DESCRIPTORS
Study Design Type	"transcription profiling by array"
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STUDY PUBLICATIONS
Study PubMed ID	"17557906"
Study Publication DOI	"10.1093/toxsci/kfm156"
Study Publication Author List	"Fielden MR, Brennan R, Gollub J"
Study Publication Title	"A gene expression biomarker provides early prediction and mechanistic assessment of hepatic tumor induction by nongenotoxic chemicals."
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STUDY FACTORS
Study Factor Name	"treatment duration"	"treatment compound"	"administration route"	"dose"	"experiment date"	"solvent"
Study Factor Type	"treatment duration"	"treatment compound"	"administration route"	"dose"	"experiment date"	"solvent"
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STUDY ASSAYS
Study Assay File Name	"a_E-GEOD-8251_GeneChip_assay.txt"
Study Assay Measurement Type	"transcription profiling"
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Study Assay Technology Type	"DNA microarray"
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STUDY PROTOCOLS
Study Protocol Name	"P-GSE8251-3"	"P-GSE8251-2"	"P-GSE8251-4"	"P-GSE8251-5"	"P-GSE8251-6"	"P-GSE8251-7"	"P-GSE8251-8"	"P-GSE8251-1"
Study Protocol Type	"grow"	"specified_biomaterial_action"	"nucleic_acid_extraction"	"labeling"	"hybridization"	"image_acquisition"	"feature_extraction"	"bioassay_data_transformation"
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Study Protocol Description	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44. Sprague-Dawley (Crl:CD(SD)|GS BR) rats (aged 6-8 weeks and weighing 200 - 260 g), were purchased from Charles River Laboratories (Wilmington, MA). They were housed in plastic cages for 1 week for acclimation to the laboratory environment of a ventilated room (temperature, 22°C ± 3°C; humidity, 30 - 70%; light/dark cycle, 12 h d-1, 6:00am - 6:00pm) until use. Certified Rodent Diet #5002 (PMI Feeds Inc.) and chlorinated tap water was available ad libitum."	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44. Compounds were administered via the route of administration (ROA) that corresponds to that by which humans receive the drug. Compounds were typically administered orally (PO) (83%), intravenously (IV) (9.4%), subcutaneously (SC) (5.7%), or intraperitoneally (IP) (2%). If the compound is a toxicant or a biochemical standard, it was administered orally. For oral dosing, the vehicle choice largely depends on the solubility of the compound in water. Water-soluble compounds were administered in water. Insoluble compounds are administered in either corn oil or 0.5% carboxymethylcellulose (CMC) using the best literature recommendation as a guide. IV administered compounds were usually dissolved in saline and SC administered compounds in corn oil. The highest dose the rats receive is the Maximum Tolerated Dose (MTD). The dosing of animals was staggered based on intended harvest order to ensure that sacrifice occurred within 30 minutes of the recorded time point. Dosing frequency was as described in Sample_characteristics for the particular treatment."	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44. Following sacrifice, 6 mm disposable biopsy punches (#REF 33-36 Miltex, Inc. Bethpage, NY) were used to obtain tissue samples of approximately 100 mg. Samples were placed in cryogenic tubes and snap frozen. Poly A(+)-RNA from tissue samples was isolated using the MagNA Pure LC robot (Roche, Basel, Switzerland) in combination with the MagNA Pure LC mRNA Isolation kit I and II (Roche, Basel, Switzerland) for cells (see supplemental methods section) and tissues, respectively. Tissue samples were completely homogenized directly from ~100 mg punches stored on dry ice prior to application of lysis buffer to a final concentration of 65 mg tissue per ml of buffer. After complete homogenization, using disposable Omni Tip Disposable Generator Probes (Omni Inc, Warrenton, VA) and before loading of the samples into the 32-well MagNA Pure plate, the samples were drawn up 5-6 times through a 20-gauge needle attached to a 3-ml syringe to ensure any tissue pieces or clumps were removed from the lysate prior to robotic processing. Tissue sample processing was performed in duplicate wells (loading 150 ul of homogenized sample to each well) of the MagNA Pure LC, which is programmed to extract mRNA using the oligo-dT selection method into a final elution volume of 100 ul. Poly A(+) RNA sample concentration was performed manually using a standard ethanol precipitation protocol in the presence of glycogen (50 ug/ml). After precipitation the final purified RNA sample was resuspended in 7 µl DEPC-H2O and quantified using a Ribogreen high-range assay (Molecular Probes) on the Wallac Victor2 Fluorometer (Perkin-Elmer, Fremont, CA). Additionally, the integrity of each RNA sample was determined, by comparison to historical standards (no gross degradation should be visible as suggested by clear 18S and 28S peaks on top of a hump of complex RNA, with lower amounts of RNA below 18S than under and above the 18S peak), using the Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA) in combination with the RNA 6000 Nano Lab Chip kit (Agilent Technologies)."	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44. The methods used for cRNA preparation (cDNA synthesis, cRNA preparation, and cRNA purification) are essentially as described in the CodeLink™ manual v2.1 as supplied by Amersham Biosciences (Piscataway, NJ) using the Qiagen BioRobot 9604 (Valencia, CA). cDNA synthesis, cRNA preparation, and cRNA purification were completely processed in a 96-well format using the automated Qiagen BioRobot 9604 procedure. 0.6 – 20 µg of enriched RNA from different tissue sources were added to a reaction mixture in a final volume of 12 µl, containing bacterial control RNA (1.5pg FixA, 5pgYjeK, 5pg AraB, 15pg EntF, 50pg FixB, 150pg HisB, 500pg LeuB, 1500pg gnd) and 1.0 µl of 100 pmol/µl T7-(dT)24 oligonucleotide primer (Proligo, Boulder, CO). The T7-(dT)24 oligonucleotide primer used, is an HPLC purified 63-mer with the sequence : 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3'. The mixture was incubated for 10 min at 70°C and chilled on ice. On ice, 4 ul of 5x first-strand buffer, 2 ul 0.1 M DTT, 1 ul of 10 mM dNTP mix and 1 ul Superscript™II RNaseH-reverse transcriptase (200 U/ul) were added to the mixture to make a final volume of 20 ul. The mixture was incubated for 1 hr at 37°C. Second-strand cDNA was synthesized in a volume of 150 ul, containing 92 ul nuclease-free water, 30 ul of 5x second-strand buffer, 3 ul of 10 mM dNTP mix, 4 ul of Escherichia coli DNA polymerase I (10 U/µl) and 1 ul of RNase H (2 U/ul) for 2 hr at 16°C. The cDNA was purified using a Qiagen QIAquick purification kit, and completely dried down using a Speed-Vac concentrator (45°C) for 2 hr. The dried product was resuspended in IVT reaction mix containing 3.0 ul of nuclease-free water, 4.0 ul 10x reaction buffer, 4.0 ul 75 mM ATP, 4.0 ul 75 mM GTP, 3.0 ul 75 mM CTP, 3.0 ul 75 mM UTP, 7.5 ul 10 mM Biotin 11-CTP, 7.5 ul 10 mM Biotin 11-UTP and 4.0 ul enzyme mix. The reaction mix was incubated for 14 hr at 37°C using an MJ Research 96-well PTC-200 Thermal Cycler (MJ Research, Waltham, MA), before the cRNA was purified using a Qiagen RNeasy kit. The resulting cRNA yield was quantified using the 96-well µQuan Universal Microplate Spectrophotometer Model MQX200 (BIO-TEK Instruments Inc., Winooski, VT) at a wavelength of 260 nm. Conformance of the cRNA sample to historical size distributions (the bulk of the cRNA product should be between 500 and 3,000 bases in size) was confirmed using the Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA). Samples not near the historical norm were reprocessed starting from tissue or RNA."	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44. 12.5 ug of cRNA sample was fragmented in 40 mM Tris-acetate (TrisOAc) pH7.9, 100 mM KOAc and 31.5 mM MgOAc at 94°C for 20 min. This typically resulted in a fragmented cRNA with a size range between 100 to 200 bases. 10 ug of the fragmented cRNA was used for hybridization of each Rat-Uniset I (RU1) Expression BioArray (Amersham Biosciences, Piscataway, NJ) in a volume of 260 ul, containing 78 ul of CodeLink Hyb buffer component A and 130 ul of CodeLink Hyb buffer component B (Amersham Biosciences, Piscataway, NJ). The hybridization solution was denatured at 90°C for 5 min then chilled on ice. The sample was vortexed at maximum speed for 5 sec and centrifuged at maximum speed for 5 min before 250 ul of the solution was injected into the inlet port of the flex-hybridization chamber, and placed in a CodeLink 12-slide shaker tray. The hybridization ports were sealed with 1 cm sealing strips (Amersham Biosciences, Piscataway, NJ), and the shaker tray(s) containing the slides was loaded into a New Brunswick Innova 4080 shaking incubator, with the hybridization chambers facing up. Slides were incubated for 20 hr at 37°C, while shaking at 300 rpm. The 12-slide shaker tray was removed from the shaker, and the hybridization chamber removed from each slide. Each slide was placed into the BioArray Rack of the Parallel Processing Tool (Amersham Biosciences, Piscataway, NJ) and incubated with 0.75x TNT (0.075 M Tris-HCl, pH7.6, 0.1125 M NaCl, 0.0375 % Tween-20) at 46°C for 1hr. The BioArray Rack was moved from the TNT containing reservoir to the small reagent reservoir containing 1:500 dilution of streptavidin-Alexa 647 (Molecular Probes, Eugene, OR). The signal was developed for 30 min at room temperature, before the reaction was stopped and slides were washed four times for 5 min each in TNT buffer (0.1 M Tris-HCl, pH7.6, 0.15 M NaCl, 0.05% Tween-20) using a large reagent reservoir. The slides were rinsed in ddH2O with 0.05% Tween-20 twice for 5 sec each before they were dried by centrifugation with a Qiagen Sigma 4-15C centrifuge (Valencia, CA) using a swinging bucket rotor (2x96) for exactly 3 min at 2000rpm (acceleration at position 9 and deceleration at position 9). The dried slides were stored in light protective slide boxes at room temperature prior to scanning."	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44. The Axon GenePix Scanner (Axon Instrument, Union City, CA) was calibrated using the Calibration Slide supplied by Axon Instrument with GenePix 4.0 at 635 nm using the Calibration System. After calibration of the scanner, all processed slides were scanned with the laser set to 635 nm, the photomultiplier tube (PMT) voltage to 600 and the scan resolution to 10 microns. For consistency all slides were scanned the same day of color development, within an hour after dry spinning them and the data was analyzed using the CodeLink Expression Analysis Software version 2.2.25 (Amersham Biosciences, Piscataway, NJ)."	"See Ganter et al., J Biotechnol. 2005 Sep 29;119(3):219-44, supplemental methods. Prior to statistical computation, the spot reading data was normalized. For this purpose a new nonlinear normalization procedure, similar to the centralization approach reported in the literature (Zien et al., 2001) was developed for the CodeLink array platform. The normalization procedure uses proprietary algorithm that assumes that in general, for an array with many probes, the majority of the signal represents genes that have unchanged expressions compared to controls, with the extreme values representing the true biology of the process and not some artifact due to measurement noise. The algorithm does not make the assumption that the true mRNA abundance being measured is linearly proportional to the spot reading signal or any assumption about the error distribution of such signals or their differential. Rather, the assumption of unchanged signals representing the bulk of the signal measurements is used to center a nonlinear curve fit to a reference template. This reference template is constructed for a large set of time matched, same tissue and same vehicle, control arrays, computing a median log signal for each probe. The replicate size of this set is adequate to ensure very small random error in the ensemble signal level for each reference probe. The curve fit corrects for some array processing problems, such as partial signal saturation, and improves the overall quality of the data compared to simple linear normalization methods. Essentially the curve tracks the mode of the signal distribution for sets of genes against the expected value for that set."	"ID_REF = <br>VALUE = Normalized signal<br>RAW_SIGNAL = Raw signal value"
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STUDY CONTACTS
Study Person Last Name	"Gollub"	"Fielden"	"Brennan"	"Gollub"
Study Person First Name	"Jeremy"	"Mark"	"Richard"	"Jeremy"
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Study Person Email	"jgollub@iconixbiosciences.com"	""	""	""
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Study Person Address	"Iconix Biosciences, 325 E. Middlefield Rd., Mountain View, CA, USA"	""	""	""
Study Person Affiliation	"Iconix Biosciences"	""	""	""
Study Person Roles	"submitter"	""	""	""
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