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).
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 2
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).
