Premix ex taq 2

5 µg of total RNA was incubated with or without 3 U/µg RNase R (Epicentre Technologies, Madison, WI, USA) for 15 min at 37 °C. After treatment with RNase R, CircRNA NRIP1 levels were detected by qRT-PCR. Reverse transcriptional reaction was conducted with a PrimeScript™ RT reagent Kit (Promega, Madison, WI) or TaqMan MicroRNA Reverse Transcription Kit (Promega) according to the manufacturer’s manual. qRT-PCR reactions involved employing the Light Cycler-DNA Master SYBR Green qPCR mixture (Bio Rad, Hercules, CA) for miRNA and the Premix Ex Taq™ II (Bio Rad, Hercules, CA) for mRNA with a Biosystems 7500 fast Real-Time PCR System (Thermo, CA, USA). The qPCR amplification conditions included a 5 min denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 60 °C for 1 min. Fluorescent signals were normalized to U6 and GAPDH for miRNA and circRNA, respectively. Then the 2^^−∆∆Ct method was applied to valuate and quantify the relative expression levels. The sequences of primers were shown in the supplementary Table 1.

Total RNA from tissues was extracted using TRIzol methods (Invitrogen) and reversely transcribed into cDNA through PrimeScript™ RT Kit (Takara). SYBR® PremixEx Taq™ II and Bio‐Rad CFX system were used to analyze the expression of genes. Quantitative RT‐PCR data were determined based on cycle threshold (Ct) and normalized to internal loading control genes GAPDH. The primers sequences are listed in Table 1.