Table 3.
Evaluation of commercial qPCR master-mixes by amplicon derived standards.
| QIAGEN R2 SYBR master-mix | QIAGEN QuantiNOVA SYBR Master-mix | Bio-rad ssoadvanced SYBR Master-mix | Applied biosystems PowerUp SYBR master-mix | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| [Primer] | Transcript | E′ (%) | LOD (copies/reaction) | R2 | E′ (%) | LOD (copies/reaction) | R2 | E′ (%) | LOD (copies/reaction) | R2 | E′ (%) | LOD (copies/reaction) | R2 | Primer failure rate (%) |
| 250 nM | RPLA13a | 92.2 | 101 | 0.99 | 94.9 | 102 | 0.99 | 89.1 | 101 | 0.99 | 92.0 | 101 | 0.99 | 31.3 |
| SDHA | 107.2 | 101 | 0.99 | 102.1 | 101 | 0.99 | 94.3 | 101 | 0.99 | 94.5 | 101 | 0.99 | ||
| TBP | 86.1 | 101 | 0.99 | 94.7 | 101 | 0.99 | 90.0 | 101 | 0.99 | 89.7 | 101 | 0.99 | ||
| IFN-γ | 93.6 | 101 | 0.99 | 85.8 | 101 | 0.98 | 83.9 | 102 | 0.99 | 99.7 | 101 | 0.98 | ||
| 250 nM failure rate | 25.0% | 25.0% | 50.0% | 25.0% | ||||||||||
| 500 nM | RPLA13a | 88.8 | 101 | 0.99 | 96.9 | 101 | 0.99 | 86.0 | 102 | 0.99 | 92.7 | 101 | 0.99 | 37.5 |
| SDHA | 93.8 | 101 | 0.99 | 93.5 | 102 | 0.99 | 92.4 | 101 | 0.99 | 90.8 | 101 | 0.99 | ||
| TBP | 88.6 | 101 | 0.99 | 82.6 | 102 | 0.99 | 89.2 | 101 | 0.99 | 98.4 | 101 | 0.99 | ||
| IFN-γ | 93.5 | 101 | 0.99 | 87.1 | 102 | 0.99 | 92.4 | 101 | 0.99 | 95.2 | 101 | 0.99 | ||
| 500 nM failure rate | 50% | 50% | 50% | 0% | ||||||||||
| 750 nM | RPLA13a | 98.2 | 101 | 0.99 | 87.7 | 102 | 0.99 | 96.8 | 101 | 0.99 | 94.0 | 101 | 0.99 | 37.5 |
| SDHA | 98.0 | 101 | 0.99 | 104.2 | 101 | 0.99 | 102.1 | 101 | 0.99 | 92.3 | 102 | 0.97 | ||
| TBP | 86.7 | 102 | 0.99 | 89.2 | 101 | 0.99 | 87.2 | 101 | 0.99 | 85.5 | 102 | 0.99 | ||
| IFN-γ | 94.4 | 101 | 0.99 | 84.0 | 101 | 0.99 | 92.3 | 101 | 0.99 | 96.3 | 101 | 0.99 | ||
| 750 nM failure rate | 25.0% | 75.0% | 25.0% | 25.0% | ||||||||||
| Master-mix failure rate | 33.3% | 50.0% | 41.7% | 16.7% | ||||||||||
| Overall failure rate | 35.4% | |||||||||||||
Commerical qPCR master-mixes were evaluated in combination with three primer concentrations (250, 500, and 750 nM) with logarithmically-diluted amplicon derived standards (106 to 101 copies amplicon per reaction). Assay performance i.e., reaction efficiency (E′), limit of detection (LOD) and standard coefficient of determination (R2) determined as per MIQE guidelines. Amplicons were purified once and measured in qPCR technical triplicate. E′, Dynamic Range and R2 were calculated from the mean of the qPCR technical triplicates. Failure rate calculated as percentage outside accceptable E′ (90–110%).