After the total exosomal RNA was extracted and RNA integrity was checked according to the above method, RNA quantity and quality were measured with a Nano Drop ND-1000 spectrophotometer (Thermo, USA). Then, the RNA was digested using RNase R (Epicenter Biotechnologies, USA) and purified. Complementary DNA was acquired from reverse transcription of 500 ng RNA using PrimeScript RT reagent with gDNA Eraser (TaKaRa, Japan). Primer-BLAST was applied to design the specific divergent primers, which were used to amplify the circular transcripts by head-to-tail splicing. All primers were synthesized by Realgene (Nanjing, China). After the optimal annealing temperatures were determined, qPCR was performed on the Life Tech-ViiA7 system (Applied Biosystems, USA) using PowerUP SYBR Green Master Mix (Applied Biosystems, USA) to measure the relative expression levels of circRNAs. To reduce the experimental random error, samples were loaded in triplicate and each well was treated identically. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control, and the relative expression levels of circRNAs were calculated with 2-△△Ct method. Moreover, to guarantee the accuracy of the results, all data are represented as the means ± standard deviation (SD) of three independent experiments.

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