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| Status |
Public on Feb 25, 2013 |
| Title |
Day10 MDI repl3 vs TBT 50 nM repl3 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
Day10 MDI repl3
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| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1 (ATCC CRL-173)
agent: MDI
time: Day10
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| 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, Tributyltin Chloride (TBT-Cl, Acros Chemicals, Geel, Belgium), was added for 10 days, changing the medium every 2-3 days. TBT was dissolved in DMSO with a final maximal concentration of 0.1%. Therefore, 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. In the following experiments and results, 2 days post-confluent cells are indicated by the ‘day 0’ time point. After exposure, cells were harvested for RNA extraction at day1 and day10.
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| 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using the Qiagen RNeasy mini kit
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| Label |
Cy5
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| 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).
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| Channel 2 |
| Source name |
Day10 TBT 50 nM repl3
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1 (ATCC CRL-173)
agent: TBT
agent concentration: 50 nM
time: Day10
|
| 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, Tributyltin Chloride (TBT-Cl, Acros Chemicals, Geel, Belgium), was added for 10 days, changing the medium every 2-3 days. TBT was dissolved in DMSO with a final maximal concentration of 0.1%. Therefore, 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. In the following experiments and results, 2 days post-confluent cells are indicated by the ‘day 0’ time point. After exposure, cells were harvested for RNA extraction at day1 and 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).
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| 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. A separate v+2 A-optimal hybridization design was used for each time-point. Using this design each exposure condition is represented by three biological replicates, with each biological replicate analysed in technical duplicate while applying the dye swap principle to correct for dye bias.
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| 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.
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| Data processing |
The images were analysed 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 normalisation (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).
Since a loop design was used for this microarray design, the final contrasts between the conditions (DMSO vs MDI; DMSO vs TBT 10 nM and DMSO vs TBT 50 nM) were added in separate .txt sheets with the log2 based FC values and the adjusted p-value for each ID. The .txt files are linked as supplementary files on the Series record.
The .txt files report loess normalized log2 based fold-change Cy5/Cy3.
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| Submission date |
Oct 04, 2012 |
| Last update date |
Feb 25, 2013 |
| Contact name |
Anna Pereira-Fernandes |
| E-mail(s) |
Anna.Pereira-Fernandes@ua.ac.be
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| Organization name |
University of Antwerp
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| Department |
Biology
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| Lab |
SPHERE
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| Street address |
Groenenborgerlaan 171
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| City |
Antwerp |
| ZIP/Postal code |
2020 |
| Country |
Belgium |
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| Platform ID |
GPL11202 |
| Series (1) |
| GSE41340 |
Mechanistic analysis of the model obesogen Tributyltin in the 3T3-L1 cell line |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1015202_Day10_MDI_repl3_vs_TBT_50_nM_repl3.gpr.gz |
6.1 Mb |
(ftp)(http) |
GPR |
| Processed data are available on Series record |
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