Qpcr reagent kit

Total RNA was extracted from the liver tissue using TRIzol reagent (15596026, Thermo Fisher). Reverse transcription reactions were conducted according to the instructions for the PrimeScript reagent kit (RR047A, TaKaRa). qPCR amplification was conducted using a 25 μL reaction system with a PCR amplifier (iQ5, Bio‐Rad) according to the instructions of the qPCR reagent kit (RR820A, TaKaRa). The relative quantification of RNA, in number of folds, was calculated by the 2^−ΔΔCt method. The gene sequences were acquired from GenBank, and GAPDH was used as the internal reference. The primers were synthesized by Shanghai General Biotech Co., Ltd., (primer information is listed in Table S2).

Total RNA was extracted from cultured cells or fresh LUAD tissues with TRIzol reagent (Thermo Fisher Scientific, USA) following the manufacturer's instructions. Quality assessment and quantification of the total RNA were performed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA was separately synthesized using a commercial miRNA and mRNA reverse transcription PCR kit (Takara, Dalian, China, 638315 and Takara, Dalian, China, RR047A, respectively). All qPCR analyses were performed using a commercial qPCR reagent kit (TAKARA, RR820A, China) on a real-time PCR detection system (ABI QuantStudio 7, USA). Quantification of mRNAs and miRNAs was normalized to the levels of GAPDH and U6, respectively. All miRNA primers and mRNA primers are listed in Table S3 and Table S4, respectively. The miRNA reverse primers and U6 primers were present in the reagent kit.