ONTOLOGY SOURCE REFERENCE
Term Source Name	"CHEBI"	"EFO"	"BTO"	"NCBITAXON"	"PR"	"XCO"	"UO"	
Term Source File	"http://data.bioontology.org/ontologies/CHEBI"	"http://data.bioontology.org/ontologies/EFO"	"http://data.bioontology.org/ontologies/BTO"	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/PR"	"http://data.bioontology.org/ontologies/XCO"	"http://data.bioontology.org/ontologies/UO"	
Term Source Version	"78"	"111"	"20"	"2"	"56"	"33"	"42"	
Term Source Description	"Chemical Entities of Biological Interest Ontology"	"Experimental Factor Ontology"	"BRENDA Tissue and Enzyme Source Ontology"	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"Protein Ontology"	"Experimental Conditions Ontology"	"Units of Measurement Ontology"	
INVESTIGATION
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INVESTIGATION PUBLICATIONS
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INVESTIGATION CONTACTS
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STUDY
Study Identifier	"E-GEOD-19662"
Study Title	"Gene expression profiling of rat hepatocytes after a 24h treatment with various drugs and other chemicals. [original title: Identification of biomarkers that distinguish chemical contaminants using a gradient feature selection method]"
Study Description	"531 arrays; 108 different treatments (including aspirin, ibuprofen, indomethacin, acetaminophen) plus replicates and controls. Many of the chemicals are derivatives of the explosive TNT. [original description: There are many toxic chemicals to contaminate the world and cause harm to human and other organisms. How to quickly discriminate these compounds and characterize their potential molecular mechanism and toxicity is essential. High through put transcriptomics profiles such as microarray have been proven useful to identify biomarkers for different classification and toxicity prediction purposes. Here we aim to investigate how to use microarray to predict chemical contaminants and their possible mechanisms. In this study, we divided 105 compounds plus vehicle control into 14 compound classes. On the basis of gene expression profiles of in vitro primary cultured hepatocytes, we comprehensively compared various normalization, feature selection and classification algorithms for the classification of these 14 class compounds. We found that normalization had little effect on the averaged classification accuracy. Two support vector machine methods LibSVM and SMO had better classification performance. When feature sizes were smaller, LibSVM outperformed other classification methods. Simple logistic algorithm also performed well. At the training stage, usually the feature selection method SVM-RFE performed the best, and PCA was the poorest feature selection algorithm. But overall, SVM-RFE had the highest overfitting rate when an independent dataset used for a prediction in this case. Therefore, we developed a new feature selection algorithm called gradient method which had a pretty high training classification as well as prediction accuracy with the lowest over-fitting rate. Through the analysis of biomarkers that distinguished 14 class compounds, we found a group of genes that mainly involved in cell cycle were significantly downregulated by the metal and inflammatory compounds, but were induced by anti-microbial, cancer related drugs, pesticides, and PXR mediators. For in vitro experiment, primary cultured rat hepatocytes were treated one of 105 compounds with relative controls. At least three biological replicates were used for each unique condition. In total 531 arrays were used.]"
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Study Public Release Date	"2010-01-05"
Study File Name	"s_E-GEOD-19662_study_samples.txt"
STUDY DESIGN DESCRIPTORS
Study Design Type	"transcription profiling by array"
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STUDY PUBLICATIONS
Study PubMed ID	"21073692"
Study Publication DOI	"10.1186/1752-0509-4-153"
Study Publication Author List	"Deng Y, Johnson DR, Guan X, Ang CY, Ai J, Perkins EJ"
Study Publication Title	"In vitro gene regulatory networks predict in vivo function of liver."
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STUDY FACTORS
Study Factor Name	"treatment"	"dose"
Study Factor Type	"treatment"	"dose"
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STUDY ASSAYS
Study Assay File Name	"a_E-GEOD-19662_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-GSE19662-3"	"P-GSE19662-2"	"P-GSE19662-4"	"P-GSE19662-5"	"P-GSE19662-6"	"P-GSE19662-7"	"P-GSE19662-1"
Study Protocol Type	"growth protocol"	"sample treatment protocol"	"nucleic acid extraction protocol"	"labeling protocol"	"hybridization protocol"	"array scanning protocol"	"normalization data transformation protocol"
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Study Protocol Description	"The primary rat hepatocytes, rtNHeps (AC-2630), isolated from male Sprague Dawley and its hepatocyte culture medium (HCM), and supplements and growth factors (CC-3198) were purchased from Cambrex BioScience (Walkersville, MD). Freshly isolated hepatocytes were shipped overnight on ice and processed immediately upon receipt. These cells were seeded on BioCoat Type 1 Collagen-coated T-25 (BD356484) or T-75 (BD356485) flasks, or clear, flat-bottom, 96-well plates (BD356407) that were purchased from BD Biosciences (Palo Alto, CA). Phosphate buffered saline (PBS, 21600-010), Trypsin-EDTA (15400-054), Trypan Blue stain (15250-061), Leibovitz medium (12448-015) were obtained from GIBCO-InVitrogen (Grand Island, NY)."	"The chemicals for the entire OMICS study were purchased from various sources: Energetics from Chem Service (West Chester, PA) and SRI International (Menlo Park, CA); remaining chemicals from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Fair Lawn, NJ). The purity of all these compounds was equal or greater than 98%. Prior to testing, the compounds were made up and serially diluted in dimethyl sulfoxide, DMSO (Fisher Scientific, Fair Lawn, NJ). Metal compounds were made up and serially diluted in 0.1Fm-filtered, ultra-pure water before testing. The 24-h toxicity of each OMICS compound was first determined in the human hepatocyte cell line HepG2 using the Neutral Red cytotoxicity kit (In Vitro Toxicology Kit Tox-4) obtained from Sigma-Aldrich.Primary rtNHeps cells collected at the end of the 24h chemical exposure were immediately stored in RNA Later (7021) purchased from Ambion (Austin, TX). The primary rtN Heps cells were reconstituted in HCM supplemented with ascorbic acid, fatty acid-free bovine serum albumin, transferrin, insulin, recombinant human epidermal growth factor, hydrocortisone 21 hemisuccinate, Gentamicin sulfate, and Amphotericin B immediately upon receipt. An aliquot of the cell suspension was stained in a 0.05% Trypan Blue solution and counted under an inverted microscope. The cells were then seeded at 3 x 10^6 cells per (Type 1 collagen-coated) T-75 flask. The flasks were left in a 37°C, 5% CO2 incubator overnight to allow for cell attachment. The cells were replenished with fresh HCM and dosed in triplicate flasks with the non-toxic concentration of each compound (pre-determined in the HepG2 cells) at 1% DMSO (v/v) or with solution at 1% water (v/v). Another set of triplicate flasks were dosed with the appropriate solvent control. Hence, for every 3 chemicals and a solvent control, a total of 12 flasks were used.After dosing, the flasks were returned to the incubator and incubated for 24 hours. The cells were washed with 1x PBS and detached with 1x trypsin-EDTA. The cells were left in tryspin-EDTA no longer than 5 minutes. Freshly warmed HCM was then added to neutralize the trypsin. The cells were then transferred into a tube and centrifuged. RNA Later was added the cell pellet and stored at –20°C until genomic analysis."	"Total RNA was extracted from the primary rat hepatocyte cell pellet. Cell pellets were homogenized in the lysis buffer with FAST Prep-24 from MP at speed 6.0/s twice, each last 30s before using RNeasy kits (Qiagen). Total RNA concentrations were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity and quality of total RNA was checked on an Agilent 2100 Bioanalyzer (Palo Alto, CA). The gel-like images generated by the Bioanalyzer show that total RNAs have two bands, represent 18S and 26S RNA of mammalian RNA. Nuclease-free water (Ambion) was used to elute total RNA."	"Rat whole genome oligo arrays in the format of 4X44K were purchased from Agilent. Sample cRNA synthesis, labeling, hybridization and microarray processing were performed according to manufacturer’s protocol One-Color Microarray-Based Gene Expression Analysis (version 1.0). 1 µg of total RNA was used. The Agilent One-Color Spike-Mix (part number 5188-5282) was diluted 5000-fold and 5 µL of the diluted spike-in mix was added to 1000 ng of each of the total RNA samples prior to labeling reactions. The labeling reactions were performed using the Agilent Low RNA Input Linear Amplification Kit in the presence of cyanine 3-CTP."	"1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation."	"After washing, the arrays were scanned at PMT levels 350 using GenePix 4200AL scanner (Molecular Device Inc.),"	"The Feature extraction software (V. 9.5.1) from Agilent was used to automatically find and place microarray grids, reject outlier pixels, accurately determine feature intensities and ratios, flag outlier pixels, and calculate statistical confidences. First normalize by median perchip, then by median per gene, which means normalization by median value across all samples, then transformed to log2 based value. ID_REF =  VALUE = If the scanned intensity was less than 5.0 for a probe, it was transformed to 5. A perchip (within) array normalization was performed using 50 percentile values of all the probe values in the array. Data were subsequently log (base 2) transformed for statistical analyses."
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STUDY CONTACTS
Study Person Last Name	"Guan"	"Ai"	"Deng"	"Guan"	"Johnson"	"Ang"	"Perkins"
Study Person First Name	"Xin"	"Katherine"	"Youping"	"Xin"	"David"	"Choo"	"Edward"
Study Person Mid Initials	""	""	""	""	"R"	"Y"	"J"
Study Person Email	"xin.guan@usace.army.mil"	""	""	""	""	""	""
Study Person Phone	"601-6343022"	""	""	""	""	""	""
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Study Person Address	"EPP, USACE-ERDC, 3909 Halls Ferry Rd, Vicksburg, USA"	""	""	""	""	""	""
Study Person Affiliation	"USACE-ERDC"	""	""	""	""	""	""
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