Single-cell droplet digital PCR (sc-ddPCR)

The sc-ddPCR protocol commenced with the encapsulation of a single cell into one oil droplet and then proceeded to the PCR step with a set of primers and fluorescent probes, which were the same as those used in the TaqMan SNP genotyping assay mentioned above, using TaqMan Polymerase with a 5’ to 3’ exonuclease, which releases the fluorophore from the probe, followed by the detection of the fluorescent signal in the droplets. The PCR mixture consisted of 4 μl of resuspended cells at a concentration of 2.5 × 10⁵/ml or 1.25 × 10⁵/ml, 10 μl of 2 × ddPCR Supermix (Bio-Rad), wild-type and mutant allele-specific TaqMan probes at a concentration of 0.25 μM, primer mixtures at a concentration of 0.9 μM for the target gene, and nuclease-free water for a final volume of 20 μl. Droplets were generated using the Bio-Rad QX200 system (Bio-Rad) following the manufacturer’s instructions. The reactions were transferred to a 96-well plate (Eppendorf Corp., Hamburg, Germany) for PCR using a thermal cycler (Bio-Rad) under the following conditions: amplification was carried out at a regular ramp rate of 2.0 °C/s at 95 °C for 10 min followed by 40 cycles of 30 s at 95 °C plus 2 min at 56 °C. The final enzyme deactivation step occurred at 98 °C for 10 min. The 96-well plate was transferred to a QX200 Droplet Reader (Bio-Rad), and the number of fluorescent droplets was analyzed. Each droplet was analyzed individually using a two-color detection system (set to detect FAM and VIC). The fluorescent droplets were counted to provide an absolute quantification of target mtDNA in digital form using QuantaSoft software (Ver. 1.7.4.917, https://www.bio-rad.com/en-us/sku/1864011-quantasoft-software-regulatory-edition?ID=1864011, Bio-Rad).