Denaturing solution

All the GC specimens including tissues and blood, were obtained from patients who received surgical resection for GC at Shanghai General Hospital affiliated of Shanghai Jiaotong University. Before RNA extraction, all specimens were snap-frozen instantly and stored at −80^◦C. Blood samples (5 mL) were collected from all subjects in EDTA tubes and centrifuged at 3,000 rpm for 10 min at 4^◦C, then the plasma was cautiously collected and also keep it at −80^◦C until use. Total RNA extraction from tissues and plasma samples used TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and TRNzol A+ (TIANGEN, Beijing, China). miRNA used miRcute Serum/plasma miRNA isolation kit (TIANGEN, Beijing, China) according to the manufacturer’s protocols. After adding denaturing solution (Ambion) for normalization of the sample-to-sample variation, 1 ul of synthetic external control (1 umol/L; TIANGENN) was spiked into each sample.

For plasma and exosome samples, total RNA was isolated using the mirVana PARIS Kit (Ambion) following the manufacturer's protocol. After the addition of denaturing solution (Ambion), 5 μL synthetic C elegans miRNA cel‐miR‐39 (5 nM/L, RiboBio) was added to each sample to normalize sample‐to‐sample variation. For tissue specimens, we used TRIzol (Invitrogen) to extract total RNA in accordance with the manufacturer's instruction. The acquired total RNA was finally eluted into 100 μL of RNase‐free water and kept at −80°C until further use. The concentration and purity of total RNA was measured using a Nanodrop 2000 spectrophotometer (NanoDrop Technologies). Samples with total RNA concentration <10 ng/μL were excluded in the analysis.

Total RNA was extracted from 200μl serum or exosome using the mirVana PARIS Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. 5μl of synthetic C.elegans miR-39 (5 nM/L, RiboBio, Guangzhou, China) was added to each sample after the addition of denaturing solution (Ambion, Austin, TX, USA) for normalization. Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue samples. Finally, total RNA was dissolved in 100μl RNase-free water and kept at -80°C until further analysis. The ultraviolet spectrophotometer was applied to evaluate the concentration and purity of the extracted total RNA.

Total RNAs (including miRNAs) derived from exosomes were extracted by a MirVana Paris Kit (Ambion, Austin, TX, USA) according to the manual. Denaturing solution (Ambion, Austin, TX, USA) was added to each sample. Total RNAs were dissolved in 100 μL RNase-free water. For further measurement, samples were stored in a −80°C refrigerator. The concentration of RNAs was assessed by a Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). After that, reverse transcription was applied to synthesize cDNA using the TaqMan Micro-RNA Reverse Transcription Kit (Thermo Fisher Scientific). The expression of ex-miRNAs was quantified by qRT-qPCR analysis according to the instructions of the manufacturer. We examined the obtained cDNA by qRT-PCR with TaqMan microRNA primers specific for miR-31, miR-192, and miR-375 (Thermo Fisher Scientific Inc., Waltham, MA, USA). A 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA) was employed for amplification and evaluation for miRNAs extracted from the exosomes. The miRNA expressions were calculated by 2^−ΔΔCt relative to RNU6B (U6): ΔC_t = C_tmiRNA − C_tU6.

Total RNA from 200 μl plasma or exosomes was isolated using the mirVana PARIS Kit (Ambion, Austin, TX, USA) in accordance with the protocol. After the addition of denaturing solution (Ambion, Austin, TX, USA), 5 μl synthetic C.elegans miR-39 (5 nM/L, RiboBio, Guangzhou, China) was spiked into each sample for normalization of variation between samples. According to the manufacturer's instructions, Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue samples. Total RNA was lysed in 100 μl RNase-free water and stored at −80°C for further analysis. The concentration and purity of the total RNA was analyzed using the ultraviolet spectrophotometer.