Pierceable foil heat seal

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).

Briefly, a 20 µL reaction mix containing 1 × QX200 ddPCR EvaGreen Supermix, 200 nM forward- and reverse primers, 20 ng cDNA template, and 70 µL of Droplet Generation Oil for EvaGreen^® were transferred to separate sample- and oil wells in a DG8™ Cartridge for QX200™ Droplet Generator (BioRad, Hercules, CA, USA), before sealing with the Droplet Generator DG8™ Gasket (BioRad). The samples were converted to droplets using the QX200 Droplet Generator (BioRad). Approximately 40 µL droplets were transferred to 96-Well ddPCR Plates, Semi skirted (BioRad), and heat-sealed with Pierceable Foil Heat Seal (BioRad) in a PX1 plate sealer at 180 °C for 5 s before thermal cycling (Supplementary Table S2) according to the manufacturer’s description on the Veriti™ 96-well Thermal Cycler (Applied Biosystems). The PCR lid was heated to 105 °C, and the sample volume was set to 40 µL. The 96-well plate was analyzed on a QX200 Droplet Reader using the QuantaSoft software (Thermo Fisher Scientific).
Values from ddPCR were obtained from QuantaSoft software, and relative normalized expression was calculated in Microsoft Excel by applying Equations (1) and (2) before performing statistical analysis.
$$Relative Quantity \left(RQ\right)=\frac{Treatment group \left(\mathrm{copies}/\mathrm{ul}\right)}{Control group \left(\mathrm{copies}/\mathrm{ul}\right)}$$
$$Normalized expression \left(NE\right)=\frac{RQ of Treatment group}{RQ of reference gene}$$