Geneatlas hybridization station

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.

Total RNA (130 ng) enriched for low molecular weight RNA from each sample was labelled using the FlashTag Biotin HSR RNA Labelling Kit (Affymetrix) on the GeneAtlas Hybridization Station (Affymetrix) and subsequently, it was processed using the GeneAtlas Hybridization, Wash and Stain Kit for miRNA Array Strips (Applied Biosystems, USA) on the GeneAtlas Fluidics Station (Affymetrix) according to the manufacturer’s instructions. Array strip fluorescence intensities were finally determined using the GeneAtlas Imaging Station (Affymetrix). Raw data were processed and visualized using Partek Genomics Suite software (Partek, USA).