Sure print g3 human ge microarray 8 60k version 2

Total RNA was isolated from frozen sections of 10 esophageal cancer biopsy specimens using an RNeasy Mini Kit (Qiagen). Gene expression microarray analysis was carried out using Sure Print G3 Human GE Microarray 8 60K version 2.0 (Agilent Technologies) according to the manufacturer’s protocol. For comparison, we used microarray data of 10 esophageal cancer cell lines (similar to those we used in vitro) downloaded from a public database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63941). DNA methylation status of the specimens and cell lines was determined by pyrosequencing previously.

Total RNA was extracted from short hairpin RNA (shRNA)-mediated TIM-3 knockdown (KD) KASUMI-3 cells (by sh hepatitis A virus cellular receptor 2 [shHAVCR2]-1 and shHAVCR2-2) and scrambled control cells using Isogen2 (NIPPON GENE). Gene expression profiling was performed using SurePrint G3 human GE microarray 8 × 60 k version 2.0 (Agilent) per the protocol provided by the manufacturer. Briefly, cyanine-3–labeled complementary RNA (cRNA) was synthesized using the Low Input Quick Amp Labeling kit (Agilent), single-color, and 600 ng of cRNA from each sample was fragmented and hybridized to the array using a Gene Expression Hybridization Kit (Agilent). The array was scanned using an Agilent SureScan microarray scanner, and raw microarray data were loaded into the Gene Spring GX software (version 14.5; Agilent). In accordance with the guided workflow for Agilent single-color experiment, the normalization algorithm of 75th percentile shift was used, and the preprocessing baseline was adjusted to the median of all samples. Statistical analysis for gene set enrichment analysis was performed as described.