2.13. RNA isolation and quantitative RT‐PCR of mature miRNAs

Total RNA was isolated from exosomes, cells, plasma and tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Quantitative RT‐PCR was performed using TaqMan miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Briefly, cDNA was synthesized from 5 μl of total RNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and a stem‐loop RT primer (Applied Biosystems). Real‐time PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7300 sequence detection system (Applied Biosystems). All reactions, including the no‐template control reactions, were run in triplicate. C_T values were then determined using fixed threshold settings. To calculate the absolute expression levels of target miRNAs, a series of synthetic miRNA oligonucleotides at known concentrations were also reverse‐transcribed and amplified. The absolute amount of each miRNA was then calculated on the basis of a standard curve. Expression of the miRNAs in the cells was normalized to the level of U6 small nuclear RNA (snRNA).