Copy number analysis

Real-time quantitative PCR was performed to analyze EHF copy number in gastric cancers and control subjects as described previously.^{28, 46} The TaqMan probe for EHF was 5′-6FAM-AAC CTG CCT TTCTGC TTT TCA TCA GAC CC-TAMRA-3′, and the primers were 5′-CCT ATC TTTGCT GTG ACT TAG ATC ATT AG-3′ (forward) and 5′-CGG ATG AAT TCCCAT AAG TGA GT-3′ (reverse). The TaqMan probe and primers for β-actin were described previously.²⁸ Each sample was run in triplicate, and β-actin was run in parallel to normalize the input DNA. Standard curves were established using serial dilutions of normal leukocyte DNA. EHF amplification was defined by a copy number ≥4.