Primeview human gene expression array

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.

Cell RNA was extracted using Trizol (Invitrogen, CA, USA) and purified with the RNeasy Mini kit (Qiagen, Düsseldorf, Germany), according to the manufacturer’s instructions. Previous to microarray hybridization, RNA concentration and purity were determined using a UV2800 ultraviolet spectrophotometer (UNIC, New York, NY, USA). RNA concentrations ranged between 100 ng/ml and 1 mg/ml. A260/A280 ratio values ranged between 1.8 and 2.0.
The Affymetrix PrimeView™ Human Gene Expression Array (Affymetrix, Santa Clara, CA, USA) was used to profile differentially expressed genes in bladder TICs vs. bladder cancer cells following the manufacturer’s instructions. Briefly, extracted RNA template (1 mg) was reversely transcribed into cDNA and digested into fragments with endonucleases. These fragments were labeled with DNA labeling reagent and labeled cDNAs were hybridized to the microarray via incubation at 45°C and rotated at 60 rpm for 17 h. Following washing and staining, the arrays were scanned using a GeneChip Scanner3000 with GeneChip Operating Software.

SGC‐7901/shCon and SGC‐7901/shSTIL cells were harvested and their total RNA was extracted. After quantity and quality of RNA samples using NanoDrop 2000 in an Agilent Bioanalyzer 2100, the transcriptome profiles were assessed with the PrimeView Human Gene Expression Array (Affymetrix, USA), according to the manufacturer's instructions. Briefly, the RNA samples were reversely transcribed into cDNA that were used as the templates, followed by biotin‐labelling using the GeneChip 3'IVT Expression Kit (Affymetrix). The microarray hybridization, washing and staining were performed using GeneChip Hybridization Wash and Stain Kit (Affymetrix). The microarrays were scanned using the GeneChip Scanner 3000 7G to produce raw data. The DEGs were determined with criteria of a P < 0.05 or absolute Fold Change > 1. The potential functional pathways of DEGs were analyzed by Ingenuity Pathways Analysis software.

Three HeLa-GFP cells and three HeLa-SOX17 cells were used for microarrays. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). An Affymetrix PrimeView Human Gene Expression Array was used to investigate the changes in transcriptional profiles. The experiment was performed based on the manufacturer’s standard protocols. Genes with ≥1.5-fold change between two groups were identified as differentially expressed genes.

Total RNA was extracted using TRIzol method (Invitrogen, Carlsbad, CA), purified with RNeasy mini kit (Qiagen, Valencia, CA, USA), processed using a GeneChip Expression 3′-Amplification Reagents Kit (Affymetrix, USA), and interrogated with an Affymetrix Primeview Human Gene Expression Array. RNA was further purified using RNeasy mini kit (Qiagen, Valencia, CA, USA) according to Affymetrix introductions. For expression array analysis, total RNA (250 ng) was used to generate biotin-labeled cRNA using the GeneChip Expression 3’-Amplification Reagents Kit (Affymetrix, USA) according to the manufacturer’s protocol. The cRNA was hybridized to Affymetrix Primeview Human Gene Expression Array and scanned using an Affymetrix GeneChip array Scanner 3000 7G.