4.8. RNA Isolation, cDNA Synthesis and qPCR
LYSIS
RNA isolation of plasma small EVs was performed using the miRNeasy Serum/Plasma kit (217184, Qiagen, Venlo, NLD). Briefly, 1 μL of spike-in (UniSp2, UniSp4, UniSp5; 339390, Qiagen, Venlo, NLD) was added to 1000 μL of Qiazol lysis reagent (79306, Qiagen, Venlo, NLD). Then, 200 μL of the small EV sample was added. After vortexing and incubation for 5 min, 200 μL chloroform was added, vortexed, and incubated again. After a centrifugation step (12,000× cDNA synthesis was performed using the miRCURY LNA RT Kit (339340, Qiagen, Venlo, NLD) according to the manufacturer’s protocol. In brief, 5.5 μL RNA was mixed with 4.5 μL mastermix, including UniSp6/CelmiR39 spike in control and 12U heparinase I (P0735L, New England Biolabs, Ipswich, MA, USA), as patients received heparin during the intervention. After incubation for 60 min at 42 °C, and 5 min at 95 °C, it was resuspended in 90 μL RNAse-free HmiRNA qPCR was performed with the miRCURY LNA SYBR Green PCR kit (339345, Qiagen, Venlo, NLD) according to the manufacturer’s protocol on an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). A list of miCURY miRNA primer assays can be found in 