A quantitative real-time PCR (qRT-PCR) assay was performed to validate the miRNA expression. Total RNA was reverse transcribed using an oligo (dT) primer with a Mir-X miRNA First-Strand Synthesis kit (TaKaRa, Dalian, China). The 5’ forward primers for qRT-PCR validation of miRNAs included the entire sequence of the mature miRNAs, as suggested by the manufacturer, and the 3’ primer for qRT-PCR was supplied with the kit. U6 small nuclear RNA was used as the internal control. The primers are listed in Supplementary Table 4 . qRT-PCR was performed using SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China) and a 96-well Chromo4 Real-Time PCR system (Bio-Rad). The qRT-PCR conditions were as follows: predenaturation for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C.
Quantitative real-time PCR of miRNA target gene analysis was performed using SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China) and a 96-well Chromo4 Real-Time PCR system (Bio-Rad). GAPDH was used as an internal control gene. The qRT-PCR conditions were as follows: predenaturation for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The delta-delta Ct method was used to analyze the miRNA and mRNA expression levels.
Quantitative real-time PCR of miRNA target gene analysis was performed using SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China) and a 96-well Chromo4 Real-Time PCR system (Bio-Rad). GAPDH was used as an internal control gene. The qRT-PCR conditions were as follows: predenaturation for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The delta-delta Ct method was used to analyze the miRNA and mRNA expression levels.