Profiling Extracellular Vesicle CircRNAs

The nCounter Low RNA Input Amplification Kit (NanoString Technologies, Seattle, WA, USA) was used to retrotranscribe and pre-amplify 4 μL of EV-derived RNA in a Verity thermal cycler (Applied Biosystems) following NanoString’s guidelines. Briefly, samples were denatured at 95 °C for 10 min and hybridized for 18 h at 67 °C. Our custom-made nCounter panel (including 78 circRNAs, 6 linear reference genes and 4 mRNAs [30 (link)] was used to analyze EV-derived pre-amplified cDNA according to the manufacturer’s instructions. RCC files containing data outputted by the NanoString nCounter Flex System (NanoString Technologies) from each run were exported to the nSolver Analysis Software (Version 4.0.70, NanoString Technologies).