Table 2.
Experimental set up.
| Platform | qPCR | dPCR |
|---|---|---|
| Mastermix | TaqMan Genotyping mastermix (ThermoFisherScientific) | TaqMan Genotyping mastermix (ThermoFisherScientific) |
| Other reagents | N/A | RainDrop Stabilizer (2 μL per 50 μL rxn) |
| KRAS G12D/WT duplex assay [9] | 900 nM KRAS Forward: 5′-AGGCCTGCTGAAAATGACTGAATAT-3′ |
|
| 900 nM KRAS Reverse: 5′-GCTGTATCGTCAAGGCACTCTT-3′ |
||
| 250 nM KRAS WT Probe: 5′-[VIC]TTGGAGCTGGTGGCGT[NFQ/MGB]-3′ |
||
| 250 nM KRAS G12D Probe: 5′-[FAM]TGGAGCTGATGGCGT[NFQ/MGB]-3′ |
||
| HIV LTR-gag/PDH duplex assay | 900 nM LTR-gag Forward: 5'-GCCTCAATAAAGCTTGCCTTGA-3' | |
| 900 nM LTR-gag Reverse: 5’-GGCGCCACTGCTAGAGATTTT-3’ |
||
| 200 nM LTR-gag Probe: 5’-[FAM]TGTGACTCTGGTAACTAGAGATCCCTCAGAC[BHQ1]-3’ |
||
| 900 nM PDH Forward: 5′-TGAAAGTTATACAAAATTGAGGTCACTGTT-3′ |
||
| 900 nM PDH Revers: 5′-TCCACAGCCCTCGACTAACC-3′ |
||
| 200 nM PDH Probe: 5′-[VIC]CCCCCAGATACACTTAAGGGA[MGB]-3′ |
||
| Oligonucleotide purification method | HPLC | |
| Sample volume | 20 μL | 22.5 μL |
| Total reaction volume prepared | 50 μL | 55 μL |
| Consumable | 96-well plate | RainDrop source chip |
| Reaction volume loaded | 50 μL | 50 μL |
| Partition volume | N/A | 5 pL |
| Partition number | N/A | Up to 10 million |
| Instrumentation | ABI 7900HT (ThermoFisherScientific) | Droplets generated: RainDrop Source Instrument (RainDance). Thermal cycling: DNA Engine Tetrad (Bio-rad). Droplets read: RainDrop Sense instrument (Instrument Control Software v2.1.3.11157) (RainDance) |
| KRAS Cycling Parameters | 95 °C for 15 min, followed by 45 cycles of 94 °C for 60 s and 64 °C for 60 s | 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 64 °C for 60 s, then 98 °C for 10 min, 12 °C for 15 min, and 4 °C hold |
| HIV/PDH Cycling Parameters | 95 °C for 15 min, followed by 45 cycles of 94 °C for 60 s and 60 °C for 60 s | 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 60 s, then 98 °C for 10 min, 12 °C for 10 min, and 4 °C hold |
| Analysis software | SDS v2.4 (ThermoFisherScientific) | RainDrop Analyst II software (1.0.0.520) |
| Analysis parameters | Auto baseline setting, thresholds set manually and applied to all samples within an experiment | Droplets classified independently using polygonal gates, which were then universally applied across all samples within an experiment |