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Sample GSM1375718 Query DataSets for GSM1375718
Status Public on May 18, 2015
Title Wy_24h_Exp3
Sample type RNA
 
Source name Wy_24h_Exp3
Organism Mus musculus
Characteristics tissue source: liver
cell type: primary hepatocytes
studytype: Single Dose Toxicity
assaytype: ex vivo
strain: C57BL/6
compound: Wy-14,643
dose: 300
doseunit: µM
doseduration: 24
durationunit: h
dosefrequency: once
vehicle: DMSO
route: medium
Treatment protocol After a recovery period of 40-42h, the Dulbecco's Modified Eagle's (Gibco BRL, Breda, The Netherlands) culture medium was replaced by culture medium containing either one of the 6 compounds or a vehicle control (0.5% of DMSO or PBS). All chemicals were obtained through Sigma-Aldrich, except for TCDD (Cerilliant). A dose causing minimal cytotoxicity after 24h of exposure thus resulting in ±80% viability, was established by the MTT assay (Mosmann, 1983). Next, cells were treated with an IC20-24h dose of aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), cisplatin (CisPl), 2,3,7,8-tetrachloordibenzodioxine (TCDD), cyclosporin A (CsA) or Wy-14,643 (Wy) (as shown in Table 1) for 24 or 48 h before being harvested. Independent biological experiments (at least in triplicate) with hepatocytes from different mice were obtained for every time point.
Growth protocol PMH were isolated from adult male C57BL/6 mice using a perfusion method and cultured between a collagen-collagen sandwich formation in two six wells plates as described by Mathijs et al., 2010.
Extracted molecule total RNA
Extraction protocol At the end of treatment the medium was removed and PMH were harvested in Qiazol (QIAGEN Benelux B.V., Venlo, The Netherlands). Total RNA was isolated using a miRNeasy Mini Kit (QIAGEN Benelux B.V., Venlo, The Netherlands) according to the manufacturer’s protocol and followed by DNase I (Qiagen, Inc) treatment. Following purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies Netherlands B.V., Amstelveen, The Netherlands). Only RNA samples which showed clear 18S and 28S peaks and with an RNA integrity number (RIN) higher than 8 were used. Samples were stored at -80ºC until RNA hybridization.
Label Biotin
Label protocol High-density oligonucleotide GeneChips from Affymetrix were used to measure gene expression levels (Mouse Genome 430 2.0 array (45101 probes)). Targets for these arrays were prepared from 250 ng of total RNA by means of the GeneChip 3’ IVT Express Kit according to the Affymetrix protocol (Affymetrix UK Ltd, High Wycombe, UK).
 
Hybridization protocol Amplified, biotinylated and fragmented targets were then hybridized on to the arrays. After hybridization, arrays were washed and stained using an Affymetrix fluidics station and scanned by use of an Affymetrix GeneArray scanner. A total of 60 RNA samples was prepared and analyzed on GeneChip arrays (treated and control samples from each time point were at least in triplicate). Normalization quality controls, including scaling factors, average intensities, present calls, background intensities, noise, and raw Q values, appeared to be within acceptable limits for all chips. Hybridization controls BioB, BioC, BioD, and CreX were called present on all chips and yielded the expected increases in intensities.
Scan protocol standard Affymetrix protocol
Data processing All raw datasets from generated CEL files were first imported into R2.11.0 and run through an in-house quality control pipeline (available through ArrayAnalysis.org (Eijssen et al, 2013)) and then used for statistical analysis. R package is freely available for academic use from the Comprehensive R Archive Network (http://www.cran.r-project.org/). The probe sets were re-annotated by use of custom Cell Definition Files (CDF) (version 12.1.0. http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/17.0.0/entrezg.asp). This resulted in a list of 16,331 unique gene IDs. After re-annotation, data were normalized by Robust Multichip Average (RMA) normalization and log2 transformed using the R package “affy”. Low expression genes were filtered out by using a log[intensity cutoff] >5. This cutoff was based on the 1st quartile of the distribution of average intensities of all reporters.
 
Submission date Apr 28, 2014
Last update date May 19, 2015
Contact name Linda Rieswijk
E-mail(s) linda.rieswijk@maastrichtuniversity.nl
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 50
City Maastricht
ZIP/Postal code 6229 ER
Country Netherlands
 
Platform ID GPL18615
Series (4)
GSE57129 Evaluating microRNA profiles reveals discriminative responses following genotoxic or non-genotoxic carcinogen exposure in primary mouse hepatocytes [Affymetrix]
GSE57132 Evaluating mRNA and microRNA profiles reveals discriminative and compound-specific responses following genotoxic or non-genotoxic carcinogen exposure in primary mouse hepatocytes
GSE72081 Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity (mRNA)

Data table header descriptions
ID_REF
VALUE RMA normalized intensity signals

Data table
ID_REF VALUE
11287_at 9.88440999
11303_at 7.792454627
11306_at 8.362984725
11308_at 9.05975844
11350_at 6.067463334
11352_at 7.052960464
11363_at 12.38238348
11364_at 12.39562465
11370_at 10.86230617
11409_at 10.18036221
11416_at 8.993549982
11425_at 9.711980377
11426_at 6.600047992
11428_at 10.61760885
11429_at 10.4772823
11430_at 11.77085552
11431_at 7.412615234
11432_at 7.460842581
11433_at 9.994907673
11435_at 4.365341738

Total number of rows: 11029

Table truncated, full table size 228 Kbytes.




Supplementary file Size Download File type/resource
GSM1375718_19_Mouse430_2_.CEL.gz 3.5 Mb (ftp)(http) CEL
Raw data provided as supplementary file
Processed data included within Sample table

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