Transcriptome Analysis of NRAS Knockdown

mRNA expression in siRNA- (siNRAS#1) or control-transfected hPheo1 cells was analysed with the GeneChip HuGene ST 1.0 array, GeneChip^TM WT PLUS Reagent Kit, Fluidics Station 450/250, GeneChip^® Hybridization Wash and Stain Kit, and GeneChip^® Scanner 3000 (Affymetrix, Santa Clara, CA, USA). CEL files were quality-checked with Expression Console (Thermo Fisher; analysis settings: RMA, “gene level”), and CHP files analysed with Transcription Analysis Console (Thermo Fisher). Genes with p values < 0.05 (ANOVA), FDR-corrected p values < 0.25, and fold change < −1.5 or >1.5 were considered significantly regulated genes. Gene set overlaps were computed using the Molecular Signatures database webpage (MSigDB v6.2) [27 (link)] for the “Hallmark” [28 (link)] and “C2: Canonical pathways” [27 (link)] gene sets, with p values calculated from the hypergeometric distribution and corrected for multiple testing with the Benjamini and Hochberg procedure. Heatmaps displaying the expression of genes of interest were generated with Wolfram Mathematica version 11.1 (Wolfram Research, Champaign, IL, USA).