Quantification of circRNA and miRNA

The total RNA was isolated using Trizol reagent (Takara, Dalian, China, Cat no. 9108). RNase R treatment was processed at 37°C with RNase R (Epicenter, WI, USA, Cat no. RNR07250). Complementary DNA (cDNA) was then directly synthesized and reversed with PrimeScript RT Master Mix (Takara, Dalian, China, Cat no. RR036A). qRT-PCR was performed using the SYBR kit (Takara, Dalian, China, Cat no. RR820A) and analyzed via a StepOne Plus system (Life Technologies, Carlsbad, CA) [16 (link)]. GAPDH and U6 were used as control genes for circRNA and miRNA, respectively. The fold changes were calculated through relative quantification (2^−ΔΔCt). The primer sequences are provided in Table S2.