Table 1.
Characteristics of the probe-based multiplex reactions using standard curves of circular pCR2.1_HBB_N and/or pCR2.1_HBB_A 1-insert (1i) constructs separated (simplex) or together in 1:1 molar ratio (duplex) or of different conformations (circular, linear and fragmented) of the pCR2.1_HBB_N_A 2-insert (2i) construct, and of SYBR Green reactions using serial dilutions of cDNA derived from a cell culture pool (n = 6) of induced differentiated (day-3) patient-derived CD34+ cells.
| Standard Curve (SC) Composition | SC Equation | R2 | Efficiency |
|---|---|---|---|
| pCR2.1_HBB_N (Circular 1i simplex SC) | Y = −4.673 × X + 45.49 | 0.9927 | 63.28 |
| pCR2.1_HBB_A (Circular 1i simplex SC) | Y = −4.76 × X + 47.61 | 0.9976 | 62.21 |
| pCR2.1_HBB_N (Circular 1i duplex SC) | Y = −4.321 × X + 44.73 | 0.9922 | 70.38 |
| pCR2.1_HBB_A (Circular 1i duplex SC) | Y = −4.424 × X + 47.34 | 0.9872 | 68.28 |
| pCR2.1_HBB_N (Circular2i SC) | Y = −4.775 × X + 47.12 | 0.9894 | 61.97 |
| pCR2.1_HBB_A (Circular 2i SC) | Y = −5.095 × X + 50.16 | 0.9903 | 57.13 |
| pCR2.1_HBB_N (Linear 2i SC) | Y = −5.074 × X + 47.16 | 0.9972 | 57.43 |
| pCR2.1_HBB_A (Linear 2i SC) | Y = −5.275 × X + 49.16 | 0.9934 | 54.73 |
| pCR2.1_HBB_N (Fragmented 2i SC) | Y = −3.567 × X + 35.46 | 0.9935 | 90.70 |
| pCR2.1_HBB_A (Fragmented 2i SC) | Y = −3.74 × X + 39.41 | 0.9911 | 85.09 |
| Normal HBB cDNA sample dilution | Y = −3.388 × X + 23.5 | 0.9954 | 97.31 |
| Abnormal HBB cDNA sample dilution | Y = −3.483 × X + 24.56 | 0.9938 | 93.69 |
| Total HBB cDNA sample dilution | Y = −3.576 × X + 22.82 | 0.9982 | 90.39 |
| HBA cDNA sample dilution | Y = −3.454 × X + 16.91 | 0.9995 | 94.77 |
Y—threshold cycle, X—log of target quantity.