2.19. Real-time PCR of STAT3

The total RNA of the tumor tissues was extracted with Trizol (Takara, Dalian, China) following the manufacturer's protocol. The reverse transcription was achieved using the Prime Script RT reagent kit (Takara). The primers used for STAT3 and the β-actin gene were as follows: STAT3 forward, 5′-GCTA CAGCAGCTTGACACACG-3′ and STAT3 reverse, 5′-G TGGCATGTGATTCTTTGCTGGC-3’; β-actin forward, 5′- AGAAAATCTGGCACCACACC-3′ and β-actin reverse, 5′-TAGCACAGCCTGGATAGCAA-3’. β-actin was used as an endogenous control. The PCR set cycle conditions were 98 °C for 5 min, 98 °C 10 s, 50 °C for 15 s, 72 °C for 30 s, after which the relative STAT3 expression was estimated by qRT-PCR (Roche, Germany) and obtained based on the 2-ΔΔCt method (Schmittgen and Livak, 2008).