4.13. Gene Expression and Pathway Analysis

Total RNA was extracted while using Trizol reagent (Invitrogen/Thermo Fisher Scientific) and purified using RNeasy mini kit (QIAGEN). The RNA quality examined for OD260/280 greater than 1.8 using Nanodrop and RNA integrity number values greater than 7.0 using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Genome-wide gene expression analysis was performed while using the Affymetrix Mouse Clariom S Assay (Affymetrix, Santa Clara, CA, USA), including 22,100 well-annotated genes, by the Microarray Core Laboratory of the National Health Research Institutes. Microarray analysis was conducted and followed the standard Affymetrix procedures. The hybridized slides were scanned by Affymetrix Gene Chip scanner 3000 7G system (Affymetrix, Santa Clara, CA, USA). Raw data were generated while using Partek Genomics Suite version 7 software (St Louis, MI, USA). Principal component analysis (PCA) and heatmap of EMT signature genes were generated using Python PCA package and R gplots package, respectively. Pathway analysis was performed using Gene set enrichment analysis and Hallmark pathway collection, as previously described [54,55].