Microarray

Total RNA was isolated from exosomes using TRIzol reagent (Invitrogen, USA). The RNA quality was determined by ND-2000 NanoDrop (Thermo Fisher Scientific, USA). The adapter ligation, cDNA synthesis, PCR, and RNA libraries construction were performed by TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) according to the manufacturer’s instructions. Microarray assay was performed as Previously described.^30,31 Next, the samples were sent and processed in BGI Company (Beijing, China). Briefly, SurePrint Human miRNA Microarrays kit (Agilent, USA) was applied for labeling and hybridization according to the manufacturer’s instructions. The miRNA expression profile was analyzed by using the Agilent Human 8×60 K. The experiments had at least 3 repeats. The differential expression of miRNAs was defined as those with larger or less than 2-fold change meanwhile P < 0.05. Raw data were normalized by the Quantile algorithm in R software. Statistic differences were calculated using the t-test.