Cells were seeded into 35mm Petri dishes at a density of 10^5 cells/dish and were typically at 70% confluency at the time of exposure to CS. Three replicate dishes were treated for each condition, and each replicate was analyzed using a separate microarray (i.e., the RNA from the dishes was not pooled). The cell culture medium was replaced with 37°C Dulbecco’s PBS (D-PBS) containing calcium and magnesium (Gibco/Invitrogen) for the smoke exposure. The covers were removed from the Petri dishes and they were placed in a smoke exposure chamber designed to deliver a consistent dose of diluted smoke. Smoke was generated under Federal Trade Commission smoking conditions (35 ± 0.3cc puff, one puff every 60 seconds, 2-second puff duration with none of the ventilation holes blocked) using a KC 5 Port Smoker (KC Automation, Richmond, VA)
Growth protocol
NHBE cells from nonsmoking, nondiabetic donors were purchased from Cambrex Corporation (Walkersville, MD). Cells were cultured in complete Bronchial Epithelial Cell Growth Medium, prepared by supplementing Bronchial Epithelial Basal Medium with retinoic acid, epidermal growth factor, epinephrine, transferrin, T3, insulin, hydrocortisone, antimicrobial agents and bovine pituitary extract by addition of SingleQuots,TM (Cambrex Corporation).
Extracted molecule
total RNA
Extraction protocol
NHBE cell lysates were homogenized using a QIAshredder spin column and RNA extracted using Qiagen RNeasy spin columns according to the manufacturer’s protocol
Label
biotin
Label protocol
Two micrograms of total RNA was reverse transcribed into double stranded cDNA using a oligo(dT)/T7 promotor chimeric primer, and in vitro transcribed using reagents provided by Affymetrix
Hybridization protocol
Fragmented biotin-labeled cRNA at a concentration of 50 ng/ul was hybridized to Affymetrix Human Genome U133 Plus 2.0 GeneChip® expression arrays according to the manufacturer’s recommendations for a minimum of sixteen hours. Post-hybridization washing and staining was performed on the GeneChip® Fluidics Station 450
Scan protocol
Arrays were scanned with the GeneChip® Scanner 3000 7G, under the control of the Affymetrix GeneChip® Operating Software (GCOS).
