Qx200 droplet reader instrument

RNA (1 ng) was reverse transcribed into cDNA using the miScript II RT kit (Qiagen) following manufacturer’s protocol. Resulting cDNA was diluted 1:8 with DNase-free water and used as a template for droplet digital PCR (ddPCR). The QX200 ddPCR system from Bio-Rad Technologies was used to measure the concentration (copies/µL) of specific miRNA as previously described^{23 (link),26 (link)}. Briefly, a volume of 5.5 µL of diluted cDNA was added to 11 µL of QX200 EvaGreen 2 × Supermix, 2.2 µL of forward primer, 1.1 µL of miScript Universal primer and 2.2 µL of nuclease-free water. Droplets were generated by mixing 20 µL reactions with 70 µL of QX200 droplet generation oil for Eva Green on a QX200 droplet generator instrument (Bio-Rad). Cycling protocol consisted of an initial 95 °C for 5 min, then 44 cycles of 95 °C for 30 s, 56 °C for 1 min and 72 °C for 2 min, then 5 min at 4 °C and 5 min at 90 °C. Ramp speed was set to 2 °C/s. Droplet fluorescence was detected on a Qx200 Droplet Reader instrument (Bio-Rad). A minimum of 10,000 droplets needed to be detected for the sample to be valid for analysis. miScript Primer Assays (Qiagen) were used for all miRNA of interest. To account for any pipetting error, samples were run in duplicate and the average copies/µL of both wells calculated. Data are expressed as the number of miRNA copies per RNA input.