Table 2.
Formulation of the simplex and multiplex RT-PCR reagent mixes.
| Reagents | Unit | Conventional RT-qPCR | Droplet RT-PCR | |||
|---|---|---|---|---|---|---|
| Simplex (before optimization) | Simplex | Multiplex | IVT multiplex | Crude cell multiplex | ||
| TrisHCl, pH 8.0 | mM | 10 | 30 | 30 | 30 | 30 |
| KCl | mM | 55 | 55 | 90 | 70 | 70 |
| MgCl2 | mM | 2 | 2 | 2.5 | 2.5 | 2.5 |
| SUPERase•In RNase Inhibitor (Invitrogen) | U/μL | 1 | 1 | 1 | 1 | 1 |
| Taq polymerase (produced in Iscove lab) | U/μL | 0.1 | 0.3 | 0.6 | 0.6 | 0.6 |
| HotStart-IT Binding Protein (Thermo Scientific) | ng/μL | 510 | 510 | 950 | 950 | 950 |
| dNTPs | mM | 0.4 | 0.4 | 0.5 | 0.5 | 0.5 |
| Forward & reverse primers (Sigma-Aldrich) | uM | 0.3 | 0.9 | 0.6 | 0.6 | 0.6 |
| TaqMan MGB Probe (Applied Biosystems) | nM | 250 | 350 | 350 | 350 | 350 |
| Nonidet P-40 | % vol | 0.01 | 0.01 | 0.01 | 0.01 | 0.14 |
| Pluronic F-68 Non-ionic Surfactant (Gibco) | % vol | 0 | 0 | 0 | 0.05 | 0.2 |
| SuperScript III reverse transcriptase (Invitrogen) | U/μL | 1 | 1 | 1 | 1 | 1 |
| Bovine serum albumin (Roche) | ng/μL | 150 | 450 | 450 | 1380 | 1380 |
| DTT (Invitrogen) | mM | 1 | 1 | 1 | 1 | 1 |
| ROX passive reference dye | nM | 50 | 50 | 50 | 0 | 0 |