Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA reverse transcription was performed in strict accordance with the PrimeScript™ II 1st Strand cDNA Synthesis kit instructions. The system was: 2 µl 5X buffer, 0.5 µl RT Enzyme Mix, 0.5 µl Oligo (dT) primer, 2 µl Random 6 mers, 1 µg total RNA, and RNase free H₂O to 10 µl. The reaction conditions were: 37°C for 15 min and then 85°C for 5 min. After the reverse transcription, cDNA was collected. The SYBR Green PCR reaction system was: (Thermo Fisher Scientific, Inc.): PrimeScript 1 Step Enzyme Mix 1 µl, upstream and downstream primers 0.5 µl each, 2X 1 step buffer 12.5 µl, and cDNA 1 µl. Finally, cDNA RNase Free dH₂O was used to add up to 25 µl. The PCR reaction conditions were: 50°C for 30 min, 94°C degeneration for 2 min, 98°C degeneration for 10 sec, and 68°C for 1 min. Thirty circles were performed, and U6 was used as the internal reference. The data were analyzed using the 2^−∆∆Cq method (13). The experiment was carried out three times. The median relative expression of miRNA-146b-5p in patient tissues was selected and divided into the high expression and low expression groups.