Stem loop rt qpcr taqman microrna assays

The relative mRNA expression of circUbe3a, miR-138-5p, and RhoC was analyzed as previously described. Total RNA was extracted from cell lysates or SEVs using the TRIzol one-step method (Invitrogen, USA). The purity of the isolated RNA was determined by the optical density 260/280 ratio using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific). The isolated RNA was reverse transcribed using a MiRNA qRT-PCR Starter Kit (RiboBio, China). RT-qPCR analyses were performed using SYBR Premix Ex Taq II (TaKaRa). RNase R treatment was used for circRNA detection as previously described. Stem-loop RT-qPCR TaqMan MicroRNA assays (Life Technologies) were used to determine the miRNA amounts. GAPDH or U6 was used as the internal reference. All the primer sequences are listed in Table 1. Gene expression was quantified using the 2^-∆∆Ct method.

Total RNA was isolated from cell lysates using TRIzol reagent (Life Technologies, Carlsbad, CA). The cDNAs of mRNA and circRNA were synthesized by using PrimeScript RT Master Mix (Takara, Dalian, China) from 500 ng of RNA. Real-time PCR analyses were performed using SYBR Premix Ex Taq II (Takara). In particular, the divergent primers annealing to the distal ends of circRNA were used to determine the abundance of circRNA. For the absolute quantification of circHIPK3, the purified PCR product amplified from the cDNA corresponding to the circHIPK3 sequence was serially diluted to generate a standard curve. Stem-loop RT-qPCR TaqMan MicroRNA assays (Life Technologies) were used to detect the amount of miRNA. GAPDH or U6 was detected as the internal reference. All primer sequences were designed and synthesized by RiboBio (Guangzhou, China). The primers are listed in Table 1. Gene expression was quantified using the 2^−ΔΔCt method.