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Sample GSM1280273 Query DataSets for GSM1280273
Status Public on May 01, 2014
Title BPA repl 1 vs Reference
Sample type RNA
 
Channel 1
Source name Reference
Organism Mus musculus
Characteristics cell line: 3t3-L1 (CRL-173)
reference composition: a mixture of equal aliquots from control and exposed samples
Treatment protocol Cells were seeded in 6- well plates at a density of 200,000 cells/well and grown until confluence. Standard medium of 2 days post-confluent cells was replaced by mature medium containing 10% Heat Inactivated Foetal Bovine Serum (FBS, Life Technologies) instead of Newborn Calf Serum and the test compound were added for 10 days, changing the medium every 2-3 days. A solvent control (0.1% DMSO) was included in each experiment. As positive control, 2 days post-confluent cells were stimulated for 48h in mature medium containing a MDI hormonal cocktail (0.5 mM isobutyl methylxanthine, 0.25 µM dexamethasone and 10 µg/mL insulin) and another 8 days in mature medium containing only insulin (10 µg/mL) and changed every 3 days. After exposure, cells were harvested for RNA extraction at day10.
Growth protocol 3T3-L1 murine cell line (ATCC CRL-173) were cultured in 75 cm2 Nunc cell culture flasks in standard growth medium (DMEM high glucose, Gibco BRL, 31885-023) supplemented with 10% heat inactivated Newborn Calf serum, 100 U/mL Penicillin, 100 µg/mL Streptomycin (Life Technologies) and 1 mM sodium pyruvate and phenol red as pH indicator. Cells were grown in a 37°C incubator under a 5% CO2 atmosphere and cultures were routinely verified as mycoplasma free with the PCR-based VenorTM GeM Mycoplasma Detection kit (Sigma-Aldrich, Bornem, Belgium). At 70-90% confluence, cells were detached with 0.25% tryspin/EDTA during 3 min (37°C). The trypsin was neutralized with growth medium and cells were split, with a maximum of 12 passages. Every 2-3 days, medium was refreshed.
Extracted molecule total RNA
Extraction protocol Total RNA extracted using the Qiagen RNeasy mini kit
Label Cy5
Label protocol Low Input Quick Amplification Labeling Kit (LIQA, Agilent, Diegem, Belgium), according to the protocol of the manufacturers. Briefly, starting from 200 ng of total RNA, poly-A RNA was reverse transcribed using a poly dT-T7 primer. The resulting cDNA was used for one round of amplification by T7 in vitro transcription reaction in the presence of Cyanine 3-CTP or Cyanine 5-CTP. The amplified and labelled RNA samples were purified separately on an RNeasy purification column (Qiagen).
 
Channel 2
Source name BPA
Organism Mus musculus
Characteristics cell line: 3t3-L1 (CRL-173)
agent: BPA
Treatment protocol Cells were seeded in 6- well plates at a density of 200,000 cells/well and grown until confluence. Standard medium of 2 days post-confluent cells was replaced by mature medium containing 10% Heat Inactivated Foetal Bovine Serum (FBS, Life Technologies) instead of Newborn Calf Serum and the test compound were added for 10 days, changing the medium every 2-3 days. A solvent control (0.1% DMSO) was included in each experiment. As positive control, 2 days post-confluent cells were stimulated for 48h in mature medium containing a MDI hormonal cocktail (0.5 mM isobutyl methylxanthine, 0.25 µM dexamethasone and 10 µg/mL insulin) and another 8 days in mature medium containing only insulin (10 µg/mL) and changed every 3 days. After exposure, cells were harvested for RNA extraction at day10.
Growth protocol 3T3-L1 murine cell line (ATCC CRL-173) were cultured in 75 cm2 Nunc cell culture flasks in standard growth medium (DMEM high glucose, Gibco BRL, 31885-023) supplemented with 10% heat inactivated Newborn Calf serum, 100 U/mL Penicillin, 100 µg/mL Streptomycin (Life Technologies) and 1 mM sodium pyruvate and phenol red as pH indicator. Cells were grown in a 37°C incubator under a 5% CO2 atmosphere and cultures were routinely verified as mycoplasma free with the PCR-based VenorTM GeM Mycoplasma Detection kit (Sigma-Aldrich, Bornem, Belgium). At 70-90% confluence, cells were detached with 0.25% tryspin/EDTA during 3 min (37°C). The trypsin was neutralized with growth medium and cells were split, with a maximum of 12 passages. Every 2-3 days, medium was refreshed.
Extracted molecule total RNA
Extraction protocol Total RNA extracted using the Qiagen RNeasy mini kit
Label Cy3
Label protocol Low Input Quick Amplification Labeling Kit (LIQA, Agilent, Diegem, Belgium), according to the protocol of the manufacturers. Briefly, starting from 200 ng of total RNA, poly-A RNA was reverse transcribed using a poly dT-T7 primer. The resulting cDNA was used for one round of amplification by T7 in vitro transcription reaction in the presence of Cyanine 3-CTP or Cyanine 5-CTP. The amplified and labelled RNA samples were purified separately on an RNeasy purification column (Qiagen).
 
 
Hybridization protocol 825 ng of both Cy3 and Cy5 labelled cRNA were co-hybridized on a 44K Whole Rat Genome Oligo Microarray (G4131F, Agilent) for 17h at 60°C in a continuous rotation Agilent hybridization oven. Slides were subsequently washed with Agilent wash buffers and acetonitrile and finally submersed in stabilization and drying solution (Agilent Technologies, Diegem, Belgium) to prevent ozone-induced Cy5-degradation.
Scan protocol Arrays were scanned at 532 and 635 nm using a Genetix Personal 4100A confocal scanner (Axon Instruments, Union City, CA, USA) at a resolution of 5 µm. The photomultiplier tube voltage (PMT) was adjusted for each slide for the separate wavelengths to obtain an overall red/green ratio of one.
Description Sample 19
Data processing The images were analyzed using the Genepix Pro software 4.1 (Axon Instruments) for spot identification and for quantification of the fluorescent signal intensities. Statistical analysis of microarray data was performed with the R package limma. Briefly, spots for which red or green FG < BG + 2SD on all arrays were deleted before analysis (FG: medium foreground intensity; BG: average local background intensity calculated over the full microarray; SD: standard deviation of local background intensities). After background correction (backgroundCorrect, method “normexp”, offset = 50) of the median intensity data, loess normalization (normalizeWithinArrays) was applied. Linear models were fitted to intensity ratios, after which probes were ranked in order of evidence of differential expression using an empirical Bayes method. Exposure versus control contrasts were fitted to the linear models and considered significant if p<0.05 and ǀlog2FCǀ>0.75 (log2 fold change).
 
Submission date Dec 04, 2013
Last update date May 01, 2014
Contact name Anna Pereira-Fernandes
E-mail(s) Anna.Pereira-Fernandes@ua.ac.be
Organization name University of Antwerp
Department Biology
Lab SPHERE
Street address Groenenborgerlaan 171
City Antwerp
ZIP/Postal code 2020
Country Belgium
 
Platform ID GPL11202
Series (1)
GSE53004 Toxicogenomics in the 3T3-L1 cell line, a new approach for screening of obesogenic compounds

Data table header descriptions
ID_REF
VALUE loess normalized log2 based fold-change Cy5/Cy3

Data table
ID_REF VALUE
A_51_P100034 0.112556852
A_51_P100289 0.239883983
A_51_P100768 0.117077128
A_51_P100991 -0.110836379
A_51_P101137 -0.300579951
A_51_P101573 -0.026455715
A_51_P101635 -0.175533661
A_51_P101765 -0.057195903
A_51_P101858 -0.029881324
A_51_P102106 -0.017058081
A_51_P102421 0.087639504
A_51_P102459 0.071538585
A_51_P102883 0.008689479
A_51_P103209 0.059366316
A_51_P103222 -0.015660156
A_51_P103397 -0.025374676
A_51_P103509 -0.1300402
A_51_P103594 0.024864742
A_51_P103865 0.060373836
A_51_P104392 -0.015877967

Total number of rows: 23460

Table truncated, full table size 595 Kbytes.




Supplementary file Size Download File type/resource
GSM1280273_Array_8.3.gpr.gz 6.3 Mb (ftp)(http) GPR
Processed data included within Sample table
Processed data are available on Series record

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