In a custom-built smoke exposure chamber, NHBE cells were exposed to 15min FTC smoking with each 35mL puff diluted into 500cc air. Following exposure the medium was replaced with fresh and the cells placed in a 37C incubator for the times indicated.
Growth protocol
NHBE 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
total RNA was extracted using Qiagen Rneasy kits according to manufacturer's instructions
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 (Santa Clara, CA).
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).
