Gene Expression Profiling of Airway and Distal Lung Fibroblasts

Fresh lung tissue specimens from distal lungs (n=3) and airways (n=3) were obtained through bronchoscopy, and primary fibroblasts were isolated and cultured. IRB approval was obtained from the University of Pittsburgh Institutional Review Board. Total RNA was isolated from lung tissue using Qiazol (QIAGEN) and purified using the miRNeasy Mini kit (QIAGEN). RNA quantity and quality was assessed using Agilent Nanodrop and Agilent 2100 Bioanalyzer, respectively. 500 ng of total RNA meeting was Cy3 labeled using the Agilent Low RNA Input Linear Amplification Kit PLUS (One-Color). After purification and fragmentation, aliquots of each sample were hybridized to an Agilent Whole Human Genome 4 X 44K microarray, sequentially washed, and scanned on an Agilent DNA microarray scanner.