Quantifying miRNA Expression in GC Tissues

Following the indicated treatments, total RNA was extracted from human GC tissues and cultured cells using TRIzol reagent (Thermo Fisher Scientific) and was then reverse-transcribed to cDNA with a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Otsu, Japan). Real-time quantitative PCR (RT–qPCR) was performed in an ABI Step One Plus system (Applied Biosystems, Foster City, CA, USA) using SYBR Green qPCR SuperMix-UDG with ROX. All the primers were synthesized by GenePharma (Shanghai, China). All the expression data were analyzed via the 2− ΔΔCT method [19 (link)]. The U6 levels were used to normalize the miRNA levels. The sequences of the primers used are as follows: miR-128-3p, forward 5ʹAACGACATCACAGTGAACCG-3ʹ, reverse 5ʹ-CAGAGCAGGGTCCGAGGTA-3ʹ; U6, forward 5ʹ-CGCTTCGGCAGCACATATAC-3ʹ, rev-erse 5-TTCACGAATTTGCGTGTCATC-3ʹ.