Rt primer mix

Total RNA was extracted from cells using RNA isolater (Vazyme, Nanjing, China). Complementary DNA (cDNA) for mRNA or miRNA was then synthesized with HiScript RT SuperMix (Cat # R223-01, Vazyme). MiRNA/U6 snRNA (small nuclear RNA) RT primer mix and PCR specific primer set (GenePharma, China) were used to reversely synthesize cDNA for miRNAs, detect and quantify miRNAs expression, respectively. qRT-PCR was performed with qPCR SYBR® Green Master Mix (Vazyme) on Real-Time PCR system (Applied Biosystems, USA). U6 snRNA or GAPDH was used as an endogenous control for miRNA or mRNA, respectively. 2^−∆∆Ct method was carried out to measure the relative expression levels of mRNA or miRNA. The primer sequences were mentioned in our previous study [23 (link)].

Total RNA was extracted from GC cell lines using TRIzol (TransGen Biotech, China) following the standard recommendation. The cDNA was reversely transcribed with M-MLV (Vazyme, Nanjing, China). MiRNA and U6 snRNA RT primer mix and miRNA qRT-PCR primer set (GenePharma, China) were used to detect and quantify miRNA expression. SYBR Green master mix (Vazyme) was used for qRT-PCR assay on an ABI Prism 7500 Sequence Detector (Applied Biosystems, USA). The primer sequences used were listed as below: SLC7A11 forward, 5′-TCTCCAAAGGAGGTTACCTGC-3′; SLC7A11 reverse, 5′-AGACTCCCCTCAGTAAAGTGAC-3′; GAPDH forward, 5′-TGTGGGCATCAATGGATTTGG-3′; GAPDH reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. The mRNA and miRNA levels were normalized to an internal control (GAPDH or U6 snRNA), respectively. The relative expression level was calculated using 2^−ΔΔCt method [36 (link)].