Transcriptional Profiling of α5-nAChR Knockdown in Lung Cancer

RNA was isolated from lung cancer cells transfected with scrambled siRNA or α5-nAChR -specific siRNA (three replicates each). Double stranded siRNA oligonucleotides targeting CHRNA5 and a pair of negative control siRNAs were synthesized by GenePharma (China) as previously described (Ma et al., 2014 (link)). RNA samples were analyzed by microarray expression profiling using PrimeView Human Gene Expression Array platform (Affymetrix) according to the manufacturer’s instructions (Liu et al., 2014 (link)). A total of 2.5 mg of fragmented and labeled cDNA was generated using the Affymetrix GeneChip WT Terminal Labeling and Controls Kit and hybridized onto PrimeView Human Gene Expression Array according to the manufacturer’s instructions (Affymetrix). Arrays were washed, stained, and processed using Affymetrix GeneChip Fluidics Station 450 systems after which they were imaged using Affymetrix GeneChip Scanner 3000 7G for the subsequent generation of raw data. Genes differentially expressed between A549 lung cancer cells transfected with α5-nAChR-specific siRNA compared with cells transfected with scrambled siRNA were selected on the basis of a P < 0.001. Gene and functional analysis was conducted using the commercially available software GO & Pathways Analysis according to the manufacturer’s instructions.