Humanht 12 v4 sentrix expression beadchip

Microarray experiments were conducted on HumanHT-12 v4 Sentrix Expression BeadChip (Illumina, San Diego, CA, USA). Hybridization of labeled-cRNA to BeadChip, washing, and scanning were performed according to Illumina Bead Station 500X manual. Extraction of mRNA expression data and statistical analysis of raw data were performed using software provided by the manufacturer (Illumina GenomeStudio v2011.1). Expression intensities were normalized by quantile normalization technique; and using normalized intensities, genes differentially expressed in HCT116-WT and HCT116-MT cells were determined by the integrated statistical method previously reported^{14 (link)}. Our microarray data are deposited in Gene Expression Omnibus (GSE126845). Differentially expressed genes (DEGs) were selected as those with P < 0.05 and fold change > 1.5. Functional enrichment analysis was performed using BINGO 2.3 plugin for Cytoscape software (http://www.psb.ugent.be/cbd/papers/BiNGO/Home.html), DAVID software (Database for Annotation, Visualization an Integrated Discovery, v6.7; http://david.abcc.ncifcrf.gov) to identify gene ontology (GO) biological processes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways represented by DEGs with statistical significance.

For DNA microarray hybridization, RNA was pooled by mixing equal amounts of total RNA, and biotin-labeled cRNA targets were synthesized starting from 1.5 μg of total RNA. Double-stranded cDNA synthesis was performed using the Illumina® TotalPrep RNA Amplification Kit (Illumina, San Diego, CA, USA), while biotin-UTP-labeled antisense RNA was transcribed in vitro using the Ambion MEGAscript kit (Ambion Life Technologies, Carlsbad, CA, USA). All steps of the labeling procedure were performed according to the manufacturers' protocols. Microarray experiments were conducted on the HumanHT-12 v4 Sentrix Expression BeadChip (Illumina), which contains 47,231 probes, representing 31,332 annotated genes. Hybridization of labeled cRNA to the BeadChip, washing, and scanning were performed according to the Illumina Bead Station 500× manual.

Microarray experiments were conducted using HumanHT-12 v4 Sentrix Expression BeadChip (Illumina, San Diego, CA, USA). Hybridization of labeled-cRNA to the BeadChip, washing, and scanning were performed according to the Illumina Bead Station 500G manual. Extraction of mRNA expression data and statistical analysis of raw data were performed using software provided by the manufacturer (Illumina GenomeStudio v2011.1). Expression intensities were normalized by quantile normalization. Using the normalized intensities, genes with differential expression between SW480 cells and SW620 cells were determined by a previously reported integrated statistical method [14 (link)].