Humanht 12 expression beadchip

The gene expression profiles of H292 and H292-Gef cells were determined using Illumina HumanHT-12 v3 Expression BeadChip (Illumina, Inc., San Diego, CA, USA) according to the technical manual of Macrogen (Seoul, Korea). Total RNA was extracted from cells with TRI reagent (Invitrogen, Grand Island, NY, USA) following the manufacturer’s instructions. The intensity and quantity of total RNA was assessed using a Nanodrop ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE, USA), and 0.55 μg of total RNA was used to prepare biotinylated cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX, USA). After fragmentation, 0.75 μg of labeled cRNA were hybridized to the Illumina HumanHT-12 Expression BeadChip following the manufacturer’s protocols. Arrays were scanned with the Illumina Bead Array Reader Confocal Scanner. Array data export processing and analysis was performed using Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4).

The profiles in GEO databases were selected according to our selection criteria. The key words used were “expression”, “hepatocellular carcinoma” and “Illumina HumanHT-12”. For the 94 search results, the preliminary selection criteria were as follows: 1. Homo sapiens, 2. Gene expression profile; 3. All of the data were generated by an Illumina HumanHT-12 expression beadchip; 4. The control samples were collected from adjacent nontumor liver tissues; and 5. Samples were free of other therapies except radical resection. Among the eight profiles meeting the criteria, four datasets with the most samples were chosen for further analyses. The datasets were GSE36376, GSE39791, GSE57957, and GSE87630. The original Series Matrix Files of these four messenger RNA expression profiles were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The microarray platform of the GSE36376, GSE39791, and GSE57957 datasets was GPL10558 (Illumina HumanHT-12 V4.0 expression beadchip, Illumina Inc.). The GSE87630 dataset was generated by GPL6947 (Illumina HumanHT-12 V3.0 expression beadchip, Illumina Inc.). A total of 415 HCC samples and 334 nontumor samples were included in these four datasets. The mRNA, miRNA and clinical data from TCGA-LIHC (https://cancergenome.nih.gov/) were also downloaded. The flow diagram of the study is shown in Fig. 1.