Primepcr ddpcr expert design assay kit

EV-derived mRNA was reverse-transcribed to cDNA using a Maxima H Minus Reverse Transcriptase Kit (Thermo Fisher Scientific). The EV-derived mRNA was added into a reaction solution containing 1× RT Buffer, dNTPs (0.5 mM), Random Hexamer (8 μM), Maxima H Minus Reverse Transcriptase (6.5 U μL⁻¹), and RNase inhibitor (1 U μL⁻¹). The reaction was run at 55 °C for 30 min and then 85 °C for 5 min. The cDNA generated from EV-derived mRNA was detected by the PrimePCR ddPCR Expert Design Assay Kit (dHsaEXD73338942, ROS1 rearrangements) or PrimePCR ddPCR Mutation Assay Kit (dHsaCP2000020, EGFR T790M mutation, Bio-Rad, USA) according to the manufacturer’s instructions. For ddPCR, droplets were generated within a DG8 Cartridge which was preloaded with sample (20 μL) and droplet generation oil (70 μL) for each sample. All droplets were transferred into a 96-well plate accordingly and sealed with a PX1 PCR Plate Sealer. A programmed Thermal Cycler was set at 96 °C for 10 min, followed by 40 cycles of 94 °C for 30 s and 60 °C for 60 s, and finally 98 °C for 10 min. The droplets containing amplicons were quantified with a QX200 Droplet Reader using the QuantaSoft software package.