Stocks (25mM) of freshly prepared palmitic acid (PA, 16:0), palmitoleic acid (PMA, 16:1n7), stearic acid (SA, 18:0), and oleic acid (OA, 18:1n9) were dissolved in 100% ethanol (Sigma Aldrich, MO, USA). FA stock solutions were subsequently diluted 1:100 in serum-free maintenance media containing 2% (wt/vol) BSA for a final concentration of 250 µM. Differentiated preadipocytes were treated for 48 hrs with the various FA solutions starting on day 8. The control treatment consisted of cells treated with 2% (wt/vol) BSA and a 1:100 dilution of 100% EtOH for 48 hrs.
Growth protocol
3T3-L1 preadipocytes were seeded at a density of 6.0x10^4 cells per well in six-well plates. Cells were cultured at 37°C in 5% CO2 in basic media consisting of DMEM supplemented with 5% heat-inactivated FBS and 1% penicillin-streptomycin. Differentiation was induced at 2 days post confluence (i.e. day 0) by adding a standard differentiation cocktail consisting of Dex (1 µM), IMBX (0.5 mM), and insulin (5 µg/ml) to basic media. After 2 days the differentiation media was replaced with maintenance media, which consisted of basic media supplemented with only insulin (5 µg/ml). Maintenance media was changed every 2 days for the reminder of the experiment. On day 7 FBS was removed from the media and the duration of the experiment was conducted in serum-free conditions.
Extracted molecule
total RNA
Extraction protocol
After the treatment period, total RNA was extracted from differentiated preadipocytes using the Qiagen RNeasy Mini Kit (Qiagen, Mississauga, Ontario, Canada) according to the manufacturer’s protocol. Extracted RNA was quantified using a Nanodrop (Fisher Scientific, Waltham, Massachusetts), and stored at -80oC for future analyses. RNA quality was verified using the Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Santa Clara, CA, USA), and only samples with an RNA integrity number (RIN) greater than 9.0 were used for microarray analyses.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA according to the manufacturer's instructions.
Hybridization protocol
For each sample, 100 ng of total RNA was prepared for hybridization to Affymetrix Mouse Gene 1.1 ST array strips, according to the manufacturer’s instructions. Strips were then washed and stained on the Affymetrix GeneAtlas platform.
Scan protocol
Strips were scanned on the Affymetrix GeneAtlas platform.
Description
pa3
Data processing
The data was imported into R (version 2.15.0)/Bioconductor (Biobase version 2.16.0) and normalized using the robust multichip average (RMA) implementation in the Affy package (version 1.34.0). Normalized data consists of transcript cluster IDs as identifiers.
