NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1854288 Query DataSets for GSM1854288
Status Public on Jun 28, 2016
Title 4AAF_24h_Exp2
Sample type RNA
 
Source name 4AAF_24h_Exp2
Organism Mus musculus
Characteristics studymainsource: Mouse liver
studytype: Single Dose Toxicity
assaytype: ex vivo
strain: C57Bl6
compound: 4-Acetylaminofluorene
dose: 2.5
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 21 compounds or a vehicle control (0.5% of DMSO, PBS, EtOH). Seventeen chemicals were obtained through Sigma-Aldrich, TCDD through Cerilliant, DMBA through ICN, E2 through Steraloids Inc. and PhB through Brocacef Intramuraal bv. 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 di(2-ethylhexyl)phthalate(DEHP),4-Acetylaminofluorene(4AAF),Curcumin(Cur),Phenobarbital(PhB),Reserpine(Res),7,12-Dimethylbenzanthracene (DMBA),Resorcinol(Resorcinol),Para-cresidine(pCres),Phenacetin(Phen),Diclofenac(diclo),Wy 14643(Wy),Tributyltinoxide(TBTO),Benzo[a]pyrene(BaP),8-Hydroxyquinoline [AKA 8-quinolinol](8HQ),17-b-estradiol(E2),ampicillin(AmpC),cisplatin(CisPl),Aflatoxin B1(AFB1),Cyclosporin A(CsA),2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD),Quercetin(Que) 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 before.
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.
Scan protocol After hybridization, arrays were washed and stained using an Affymetrix fluidics station and scanned by use of an Affymetrix GeneArray scanner. A total of 189 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.
Description DMSO_24h_Exp2_DL-Mouse A2
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/12.1.0/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 Aug 14, 2015
Last update date Jun 28, 2016
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 (2)
GSE72081 Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity (mRNA)
GSE72088 Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity

Data table header descriptions
ID_REF
VALUE RMA normalized intensity signals

Data table
ID_REF VALUE
100017_at 7.694622223
100019_at 7.330517956
100036521_at 8.571360754
100037258_at 9.315663266
100037278_at 5.886436005
100038347_at 4.898417864
100038371_at 3.514258611
100038392_at 5.111163839
100038398_at 5.573796099
100038566_at 5.935018779
100038743_at 3.941057496
100039495_at 5.335855838
100039684_at 5.184419551
100039795_at 6.320987441
100039864_at 10.05421882
100039968_at 7.611186678
100040049_at 4.783340059
100040220_at 13.01804688
100040297_at 6.89081618
100040377_at 5.828809341

Total number of rows: 11029

Table truncated, full table size 228 Kbytes.




Supplementary file Size Download File type/resource
GSM1854288_6J_Mouse430_2_.CEL.gz 3.4 Mb (ftp)(http) CEL
Raw data provided as supplementary file
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap