Total RNA was extracted from tissue samples using TRIzol reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), according to the manufacturer's protocol. Plasma cfRNA was extracted from 200 µl plasma using the RNeasy Serum/Plasma kit (Qiagen GmbH), according to the manufacturer's protocols. cDNA was synthesized and amplified using ImProm-II™ reverse transcriptase (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The primer sequences are indicated in Table IV. qPCR was subsequently performed using the SYBR qPCR mix (Takara Biotechnology Co., Ltd., Tokyo, Japan) with an Mx 3000p instrument (Agilent Technologies Inc., Santa Clara, CA, USA), according to the manufacturer's protocols. Amplification of the cDNA was confirmed using melting curve analysis. cDNA was reverse transcribed from 20 µl plasma cfRNA and the following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 5 min; 45 cycles of denaturation at 95°C for 10 sec and annealing and extending at 60°C for 20 sec. The relative expression level of each RNA was quantified using the 2−∆∆Cq method (19 (link)) with the 18S rRNA gene as the endogenous control for data normalization, since its expression level does not significantly differ in the plasma samples of patients with GC and healthy participants (20 (link),21 (link)).