Table 4.
Evaluation of commercial qPCR master-mixes by cDNA derived standards.
| QIAGEN R2 SYBR master-mix | QIAGEN QuantiNOVA SYBR master-mix | Bio-Rad ssoAdvanced SYBR master-mix | Applied biosystems PowerUp SYBR master-mix | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Transcript | E′ (%) | Dynamic range (cells/reaction) | R2 | E′ (%) | Dynamic range (cells/reaction) | R2 | E′ (%) | Dynamic range (cells/reaction) | R2 | E′ (%) | Dynamic range (cells/reaction) | R2 |
| RPL13a | 95.2 | 100–103 | 0.99 | 78.9 | 100–103 | 0.99 | 93.7 | 100–104 | 0.99 | 104.9 | 100–103 | 0.99 |
| SDHA | 79.9 | 100–103 | 0.99 | 85.7 | 100–103 | 0.99 | 98.3 | 100–104 | 0.99 | 121.5 | 100–103 | 0.99 |
| TBP | 134.7 | 102–104 | 0.99 | 99.1 | 102–104 | 0.99 | 95.9 | 101–104 | 0.99 | 109.7 | 102–104 | 0.99 |
| IFN–γ | 88.3 | 101–103 | 0.99 | 91.2 | 101–103 | 0.99 | 96.1 | 101–104 | 0.99 | 121.9 | 100–103 | 0.99 |
| Master-mix failure rate | 75.0% | 50.0% | 0.0% | 50.0% | ||||||||
| Overall failure rate | 43.8% | |||||||||||
Commerical qPCR master-mixes were evaluated with logarithmically diluted PBMC cDNA (104-101 cell cDNA equivalent per reaction). Assay performance, i.e., reaction efficiency (E′), limit of detection (LOD) and standard coefficient of determination (R2) determined as per MIQE guidelines. Biological replicates (n = 3) were extracted with single reverse transcription reactions per extraction, and measured in qPCR technical triplicate. RNA extractions were repeated. E′, Dynamic Range and R2 calculated from a representative technical extraction, calculated from the mean of a qPCR technical replicate. Failure rate calculated as percentage outside accceptable E′ (90–110%).