Adipocyte differentiation was initiated 2 days after reaching confluence (Day 0, D0) by growing cells in DMEM supplemented with 10% Fetal Bovine Serum, 100U/mL penicillin and 100µg/mL streptomycin, 1µM dexamethasone (Sigma, Ref. D8893-1MG), 500µM 3-isobutyl-1-methylxanthine (IBMX, Sigma, Ref. I5879) and 1µg/mL bovine insulin (Sigma, Ref. I5500). Two days later (day 2, D2), medium was replaced by DMEM supplemented with 10% FBS, 1µg/mL bovine insulin and a mix of 100U/mL penicillin and 100µg/mL streptomycin (Life Technologies). Two days later (day 4, D4), medium was replaced by DMEM supplemented with 10% FBS and subsequently renewed every other day until day 9, D9. 3T3-L1 cells were transfected by siRNAs (100nM) using INTERFERin (Polyplus Transfection, Illkirch, France, Ref. 409-10). siRNAs were: control siRNA (Dharmacon, ON-TARGETplus non-targeting, ref. D-001810-10-10,) and Paral1-siRNA (Silencer Select ref. 101167, Ambion). Differentiated 3T3-L1 adipocytes were reverse-transfected as follows: cells were washed with 1x PBS, trypsinized and suspended (5x105 for 800µL) in DMEM supplemented with 10% FBS containing 100U/mL penicillin and 100µg/mL streptomycin. Cells in suspension were transfected by siRNA (100nM) with INTERFERin (Polyplus Transfection, Ref. 409-10) in 12-well plates according to the manufacturer’s instructions.
Growth protocol
3T3-L1 cells were routinely grown at 37°C and 5% CO2 in Dulbecco’s Modified Eagle’s medium (DMEM)-high glucose (4.5g/L) containing 10% (v/v) Cosmic Calf Serum, 100U/mL penicillin and 100µg/mL streptomycin. Seeding densities were as follows: 5x104 cells/well (12-well cluster), 12.5x104 (6-well cluster) or 5x105 cells (P100 dish). Proliferation medium was renewed every other days.
Extracted molecule
total RNA
Extraction protocol
Extract-All extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated ssDNA were prepared according to the standard Affymetrix protocol from 300ng total RNA (GeneChip® WT PLUS Reagent Kit, Affymetrix).
Hybridization protocol
Following fragmentation, 5,5µg of ssDNA were hybridized for 16 hr at 45C on GeneChip Mouse Transcriptome Array 1.0 ST Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using GeneChip Scanner GCS3000.
Description
RMA expression value derived from Expression Console software; probe analysis
Data processing
Total RNA (100-300ng) from 3T3-L1 cells was processed for labelling, purification and hybridized to Affymetrix Genechip Mouse Genome 430 2.0 or to Mouse Transcriptome Array 1.0 (MTA 1.0) according to the manufacturer's protocol. Raw data were pre-processed (Expression Console, v1.4.1. Affymetrix), using the GCCN and SST algorithms and RMA background correction for MTA arrays.
