Disposable droplet generator cartridge

A half of the entire yield of extracted EV RNA was reverse-transcribed using miRCURY LNA RT kit (Qiagen) according to the manufacturer’s protocol. A total of 20 µL of PCR reaction containing 1:2 diluted cDNA, 10 µL of 2xEvaGreen Supermix (Bio-Rad) and either 1 µL of miRCURY LNA primer mix (Qiagen) or 2 µL of QuantiNova LNA primer mix (Qiagen) (Supplementary Table S1) was loaded into a disposable droplet generator cartridge (Bio-Rad). Then, 70 µL of droplet generation oil for EvaGreen was loaded in the corresponding wells and placed into a QX200 droplet generator (Bio-Rad). Once droplets were generated, they were transferred to a ddPCR clear semi-skirted 96-well plate (Bio-Rad), covered with a Pierceable Foil Heat Seal (Bio-Rad) and amplified in a T100 Thermal Cycler (Bio-Rad) under the following conditions: 95°C for 5 min; 40 cycles at 95°C for 30 s followed by specific primer annealing temperature (Supplementary Table S1); 4°C for 5 min; 90°C for 5 min and indefinite hold at 4°C. Programme was run at 2°C/sec rampage rate. Plate was read using a QX200 Droplet Reader (Bio-Rad) and results were analyzed using QuantaSoft™ Software (Bio-Rad). The optimal annealing temperature was determined for each assay by running it across a thermal gradient (50°C–60°C).

For each ddPCR assay, 9 μl cDNA sample (diluted 1:20), 10 μl 2X ddPCR supermix for Eva Green (Bio-Rad, Hercules, CA, United States), 1 μl of LNA PCR primers set was mixed (miRCURY LNA miRNAs PCR assays, Qiagen, Hilden, Germany). The mixture and 70 μl of droplet generation oil for Eva Green were respectively loaded into the sample and oil wells of a disposable droplet generator cartridge (Bio-Rad, Hercules, CA, United States). Therefore, droplets were generated by QX200 droplet generator device (Bio-Rad) and transferred to a 96 plate (Bio-Rad, Hercules, CA, United States). The cycling condition were 95°C for 5 min and 40 cycles (95°C for 30 s 58°C for 1 min) 4°C for 5 min and 90°C for 5 min. At the end of the PCR reaction, droplets were loaded in the QX200 droplet reader and analyzed using Quantasoft version 1.7.4 software (Bio-Rad, Hercules, CA, United States). A no template control (NTC) was included in every assay. miRCURY LNA miRNAs PCR assays used are: miRNA-26a, miRNA-26b, miRNA-30b, miRNA-30c (Qiagen, Hilden, Germany).

For each ddPCR assay, 3 μL cDNA sample, 10 μL 2× ddPCR supermix for probes (Bio-Rad), 1 μL 20× TaqMan miRNA probe and 6 μL RNase-free Water was added in a 20 μL reaction mixture. Then, the mixture and 70 μL droplet generation oil for probes (Bio-Rad) were respectively loaded into the sample wells and oil wells of a disposable droplet generator cartridge (Bio-Rad). After that, droplets were generated by QX200 droplet generator device (Bio-Rad) and carefully transferred to a 96-well PCR plate (Eppendorf). The cycling conditions were: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 57 °C for 1 min, and a final step at 98 °C for 10 min. At the end of the PCR reaction, droplets were read in the QX200 droplet reader and analyzed using the Quantasoft™ version 1.7.4 software (Bio-Rad). In addition, a no template control (NTC) was included in every assay. And the spike-in control miRNA was used as an internal calibrator to monitor extraction efficiency.