DACT1 mRNA expression levels were analyzed by quantitative reverse transcription–polymerase chain reaction (PCR) using total RNA extracted from 76 pairs of cancerous and normal gastric tissue samples using Trizol reagent (Thermo Fisher Scientific). Total RNA was reversely transcribed to first-strand complementary DNA using Primescript RT Reagent (Takara, Otsu, Japan). The real-time PCR primers for DACT1 were as follows: forward primer 5′-TGTGAATCCCAAGTACCAGTGT-3′ and reverse primer 5′-CCGTCAGACAAAGGAGAAACATT-3′. β-Actin was used to normalize DACT1 gene expression levels and amplified with forward primer 5′-AGAAAATCTGGCACCACACC-3′ and reverse primer 5′-TAGCACAGCCTGGATAGCAA-3′. Amplification reactions were executed in a 10 µL reaction volume containing 0.2 µL primers, 5 µL Master mix, and 100 ng complementary DNA. The cycling conditions were set at 95°C for 5 minutes, followed by 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds. Real-time PCR was carried out using FastStart Universal SYBR-Green Master (Vazyme, Nanjing, People’s Republic of China) with the StepOnePlus Real-Time PCR System in triplicate. The 2−ΔCT algorithm was used for calculating the expression of individual DACT1 relative to expression of β-actin.4 (link)