A549 cells were treated with ethanol control or resveratrol (25 mM) for 48 h. One replicate of control cells and three replicates of resveratrol treated cells were analyzed. At the 48 h time point TRIzol (1 ml) was added to each culture flask, incubated and insoluble material removed by centrifugation at 10,000 RPM for 10 min at 4°C. Chloroform (0.2 ml) was added to each sample which was shaken and incubated for phase separation. Samples were centrifuged at 10,000 RPM for 15 min at 4°C and the RNA (top aqueous phase) isolated. RNA was precipitated by mixing with isopropanol (0.8 ml) and centrifuging at 10,000 RPM for 10 min at 4°C. The RNA pellet was washed with 75% ethanol, dried and dissolved in Rnase-free water. Cleanup of the RNA was performed using an RNeasy spin column (Qiagen).
Label
Biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol at the University of Chicago Functional Genomics Facility according to the Affymetrix GeneChip Expression Analysis Manual (Santa Clara, CA). Briefly, 10 g of total RNA was used to synthesize double-stranded cDNA using the Superscript Choice System (Life Technologies). First strand cDNA synthesis was primed with a T7-(dT24) oligonucleotide. From the phase-log gel-purified cDNA, biotin-labeled anti-sense cRNA was synthesized using BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). After precipitation with 4M Lithium Chloride, 20 g of cRNA was fragmented in fragmentation buffer (40 mM Tris-Acetate, pH 8.1, 100 mM KOAc, and 30 mM MgOAc) for 35 min at 94°C
Hybridization protocol
Hybridizations were performed at the Functional Genomics Facility, University of Chicago.Following fragmentation, 12 g of fragmented cRNA was hybridized to U133 Plus 2.0 Arrays for 16 h at 45°C and 60 rpm in an Affymetrix Hybridization Oven 640. The arrays were washed and stained with streptavidin phycoerythrin in Affymetrix Fluidics Station 400 using the Affymetrix GeneChip protocol.
Scan protocol
GeneChips were scanned using the Affymetrix Agilent GeneArray Scanner.
