Cells:U-33 cells stably transfected with a PPARg2 expression construct (U-33/g2 cells) Time: 24 hrs Drug: No Rosiglitazone present
Extracted molecule
total RNA
Extraction protocol
For each experiment a fresh batch of cells was used, which was stored in liquid nitrogen. Cells were propagated for one passage and then seeded at the density of 3×105 cells/cm2. After 48h of growth, when cultures achieved about 80% confluency, cells were treated with either 1 uM Rosi or the same volume of vehicle (DMSO) for 2, 24, and 72h. RNA was isolated for each time point using the RNeasy kit (QIAGEN Inc., Valencia, CA). Replicate experiments were performed independently on a fresh batch of cells.
Label
biotin
Label protocol
RNA quality was assessed using the Agilent Model 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). For each microarray five micrograms of total RNA was processed using the Affymetrix GeneChip® one-cycle target labeling kit (Affymetrix, Inc., Santa Clara, CA) according to the protocol recommended by the manufacturer.
Hybridization protocol
The resulting biotinylated cRNA was fragmented and hybridized to the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix, Inc.). The arrays were washed and stained using the Affymetrix Model 450 Fluidics Station by the University of Iowa DNA Core Facility according to the protocols recommended by the manufacturer.
Scan protocol
The arrays were scanned using the Affymetrix Model 3000 scanner by the University of Iowa DNA Core Facility according to the protocols recommended by the manufacturer.
Description
Murine marrow-derived U-33 cells (previously referred as UAMS-33) represent a clonal cell line spontaneously immortalized in long term bone marrow cultures. To study the effect of PPARg2 on marrow mesenchymal stem cell differentiation, U-33 cells were stably transfected with either a PPARg2 expression construct (U-33/g2 cells) or an empty vector control (U-33/c cells). Several independent clones were retrieved after transfection and carefully analyzed for their phenotype. Clone 28.6 (representing U-33/g2 cells) and clone gammac2 (representing U-33/c cells) were used in the presented experiments. Cells were maintained in alphaMEM supplemented with heat-inactivated 10% FBS (Hyclone, Logan, UT), 0.5 mg/ml G418 for positive selection of transfected cells, 100 U/ml penicillin, 100 ug/ml streptomycin, and 0.25 ug/ml amphotericin (Sigma) at 37oC in a humidified atmosphere containing 5% CO2. Media and additives were purchased from Life Technologies (Gaithersburg, MD).
Data processing
The RMA method was used to adjust the background of perfect match (PM) probes, apply a quantile normalization of the corrected PM values and calculate final expression measures using the Tukey median polish algorithm.
