Quantitative Real-Time PCR Gene Expression Analysis

Stomach specimens were homogenized. Total RNA was extracted using RNAzol (Thermo Fisher Scientific, Waltham, MA, USA), and the concentration was diluted to 1 μg/μL. Thereafter, 5 μL RNA was reverse transcribed to prepare DNA. Approximately 2 μL of DNA was combined with 1 μL of the forward primer, 1 μL of the reverse primer (Table 1), and 10 μL of SYBR Green PCR Master Mix and amplified at 95 °C for 60 s, followed by 40 cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 35 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. The relative expression of each target gene was calculated by the 2^−ΔΔCt method [13 (link)].