Human clariom s assay platform

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.

A full transcriptome microarray analysis was performed using the human Clariom™ S Assay platform (Affymetrix, USA) at The Core Facility of Medical Genomics, Tomsk NRMC.
Statistical analysis. The Transcriptome Analysis Console (TAC) software (version 4.0) was used to process the results of the microarray (DEG analysis, including the construction of heat maps and determination of signaling pathways).