Isogen lysis buffer

Total RNA was isolated from cells in an ISOGEN lysis buffer (Nippon Gene, Toyama, Japan) followed by precipitation with isopropanol. Reverse transcription was carried out by SuperScript III First-Stand Synthesis System for RT-PCR (Invitrogen, CA, USA). PCR was carried out by AccuPrimeTaq DNA Polymerase System (Invitrogen, CA, USA). RT-PCR was performed within the linear range of amplification, typically 19–30 cycles, for SIM2, HIF1A, TP63, PTGS2, EGFR, ERBB2, TGFB1, ITGB3, and GAPDH. Quantitative real-time PCR was performed on HIF1A, VEGFA, HK2, PDK1, DDIT4, SLC2A, LDHA, BNIP3, NOS2, PDGFB, FGF2, NDUFA4L2, EPO, SLC16A3, MDR1, SIM2, and GAPDH by a Bio-Rad iCycler with iQSyber Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. DNA sequences of used primers are listed in Supplementary Table S3.

All esophageal squamous cell carcinoma (ESCC) specimens were provided by the National Cancer Center Hospital and National Cancer Center Hospital East, after obtaining written informed consent from each patient and approval by the National Cancer Center Institutional Review Board. Patients received definitive chemoradiotherapy (CRT) treatment: protracted infusion of 5-FU 800 to 1000 mg/m²/24 hours on days 1 to 4 and 29 to 32, a 2-hour infusion of CDDP 75 to 80 mg/m² on days 1 and 29, and concurrent radiation therapy at a dose of 50.4 to 60 Gy. After the completion of CRT, responders additionally received two courses of chemotherapy every 4 weeks. Treatment response was evaluated 8 weeks after CRT. For the biopsy samples, tumor portions (about 2 mm X 2 mm) of patients before and after (3–4 weeks) treatment were obtained under endoscopy from a margin of the tumor by exclusion of any central necrotic lesions to obtain viable cancer cells. All biopsy samples were immediately exposed to ISOGEN lysis buffer (Nippon Gene Co., Ltd., Toyama, Japan) or frozen with liquid nitrogen, and stored at -80°C until use.

Total RNA was isolated by suspending the cells in an ISOGEN lysis buffer (Nippon Gene, Toyama, Japan) followed by precipitation with isopropanol. We used Human Expression Array U95A version 2 (Affymetrix, Santa Clara, CA) for analysis of mRNA expression levels corresponding to 12,600 transcripts. The procedures were conducted according to the suppliers’ protocols. The expression value (average difference: AD) of each gene was calculated using GeneChip Analysis Suite version 4.0 software (Affymetrix). The mean of AD values in each experiment was 1000 to reliably compare variable multiple arrays. Hierarchical clustering is widely used as one of the unsupervised learning methods. Hierarchical clustering of microarray data was performed by the use of GeneSpring (Agilent Technologies Ltd., Palo Alto, CA), Microsoft EXCEL, and Cluster & TreeView software [29 (link)]. All the microarray data have been deposited in a MIAME compliant database, GEO; the accession number GSE47007. By Wilcoxon u-test (p<0.05) and a 2-fold change, 188 genes were selected as specific genes for 18 intestinal-type GCs, and 704 genes were selected as specific genes for 12 diffuse-type GCs. The results of a two-dimensional hierarchical clustering analysis of the 892 selected genes are shown.

Total RNA was isolated by suspending the cells in ISOGEN lysis buffer (Nippon Gene, Toyama, Japan) followed by precipitation with isopropanol. We performed expression analyses using Human Expression Array U95A version 2 (Affymetrix, Santa Clara, CA) according to the suppliers’ protocols . The expression value (average difference: AD) of each gene was calculated using GeneChip Analysis Suite version 4.0 software (Affymetrix). Hierarchical clustering of microarray data was performed using GeneSpring (Agilent Technologies Ltd., Palo Alto, CA), Microsoft EXCEL, and Cluster & TreeView [22 , 23 (link)]. All microarray data have been deposited in a MIAME compliant database, GEO (accession number; GSE47007). By Wilcoxon u-test (p < 0.05) and by showing a 2-fold change, genes expressed specifically in diffuse-type GC were selected [22 ].