Genechip fluidics station 450 systems

Total RNA from high-metastatic (MKN28-M and SGC7901-M) and low-metastatic (MKN28-NM and SGC7901-NM) GC cells was extracted using Cells-to-CT Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. All the RNA was quantified by a UV spectrophotometer (Beckman Coulter, Brea, CA, USA), and RNA integrity numbers were inspected by Agilent Bioanalyzer 2000 (Agilent). mRNA expression profiles for individuals were confirmed using human Clariom™ S Assay platform (Affymetrix). RNA samples were employed with the primers containing a T7 promoter and executed by reverse transcription reaction for the generation of single-stranded cDNA (ss-cDNA). 3′Adaptor was added to ss-cDNA, which was further converted to complementary RNA (cRNA) by in vitro transcription (IVT) amplification. CRNA was then converted to biotinylated double-stranded cDNA (ds-cDNA) using GeneAtlas® Hybridization Station (Affymetrix). Arrays were washed and stained using Affymetrix GeneChip Fluidics Station 450 systems, followed by the scanning with GeneChip® Scanner 3000 7G (Affymetrix). Differentially expressed genes (DEGs) (fold change ≥ 1.5 and^∗P < 0.05) were generated using R package.

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.