Table 1.
The optimized qPCR conditions for the eight candidate reference genes in T. ravennae
| Gene | Primer paira | Amplicon length (bp) | Optimal Tm (°C)b | Optimal primer concentration (mM) | cDNA dilution | R2 | E (%)c |
|---|---|---|---|---|---|---|---|
| EF1α | EF1α-qPCR-F1/EF1α-qPCR-R1 | 84 | 60.6 | 350 | 1/10–1/40 | 0.9996 | 107.4 |
| EF1α-qPCR-F1/ EF1α-qPCR-R2 | 102 | 60.6 | 350 | 1/10–1/40 | 0.9999 | 104.8 | |
| EF1α-qPCR-F2/EF1α-qPCR-R1 | 81 | 60.6 | 300 | 1/10–1/160 | 0.9994 | 110.3 | |
| EF1α-qPCR-F2/EF1α-qPCR-R2 | 99 | 60.6 | 250 | 1/10–1/160 | 0.9992 | 114.5 | |
| Ubi4 | Ubi4-F1/Ubi4-R1 | 86 | 60.6 | 350 | 1/10–1/160 | 0.9898 | 105.2 |
| Ubi4-F1/Ubi4-R2 | 88 | 60.6 | 300 | 1/20–1/160 | 0.9887 | 101.6 | |
| Ubi4-F2/Ubi4-R1 | 84 | 60.6 | 350 | 1/20–1/160 | 0.9921 | 98.6 | |
| Ubi4-F2/Ubi4-R2 | 86 | 60.6 | 350 | 1/20–1/160 | 0.9946 | 100.8 | |
| H3.3 | H3.3-qPCR-F1/H3.3-qPCR-R1 | 115 | 60.6 | 250 | 1/20–1/80 | 0.9929 | 110.2 |
| H3.3-qPCR-F1/H3.3-qPCR-R2 | 121 | 60.6 | 350 | 1/40–1/160 | 0.9992 | 96.9 | |
| H3.3-qPCR-F2/H3.3-qPCR-R1 | 108 | 60.6 | 350 | 1/10–1/40 | 0.9547 | 114.2 | |
| H3.3-qPCR-F2/H3.3-qPCR-R2 | 114 | 60.6 | 250 | 1/40–1/160 | 0.9876 | 102.9 | |
| TCTPH | TCTPH-qPCR-F1/TCTPH-qPCR-R1 | 86 | 60.6 | 350 | 1/40–1/160 | 0.9981 | 100.7 |
| TCTPH-qPCR-F1/TCTPH-qPCR-R2 | 94 | 60.6 | 350 | 1/10–1/160 | 0.9880 | 101.7 | |
| TCTPH-qPCR-F2/TCTPH-qPCR-R1 | 82 | 60.6 | 400 | 1/10–1/80 | 0.9780 | 103.4 | |
| TCTPH-qPCR-F2/TCTPH-qPCR-R2 | 90 | 60.6 | 400 | 1/10–1/80 | 0.9998 | 109.1 | |
| Aldolase | Aldolase-F1/Aldolase-R1 | 119 | 60.6 | 350 | 1/10–1/160 | 0.9975 | 103.5 |
| Aldolase-F1/Aldolase-R2 | 120 | 60.6 | 300 | 1/40–1/160 | 0.9894 | 108.1 | |
| Aldolase-F2/Aldolase-R1 | 112 | 60.6 | 400 | 1/10–1/160 | 0.9987 | 101.3 | |
| Aldolase-F2/Aldolase-R2 | 113 | 60.6 | 350 | 1/10–1/80 | 0.9944 | 102.5 | |
| BI1 | BI1-qPCR-F1/BI1-R1 | 139 | 60.6 | 400 | 1/10-1//40 | 0.9116 | 74.5 |
| BI1-qPCR-F1/BI1-R2 | 142 | 60.6 | 400 | 1/10–1/160 | 0.9924 | 100.3 | |
| BI1-qPCR-F2/ BI1-R1 | 133 | 60.6 | 400 | 1/10–1/160 | 0.9900 | 109.6 | |
| BI1-qPCR-F2/BI1-R2 | 136 | 60.6 | 400 | 1/10–1/160 | 0.9696 | 103.2 | |
| 8C | 8C-F1/8C-qPCR-R1 | 118 | 60.6 | 400 | 1/10–1/80 | 0.9988 | 104.6 |
| 8C-F1/8C-qPCR-R2 | 122 | 60.6 | 400 | 1/20–1/160 | 0.9998 | 122.4 | |
| 8C-F2/8C-qPCR-R1 | 117 | 60.6 | 400 | 1/20 –1/80 | 0.9958 | 109.9 | |
| 8C-F2/8C-qPCR-R2 | 121 | 60.6 | 400 | 1/10–1/80 | 0.9962 | 103.5 | |
| AH | AH-F1/AH-qPCR-R1 | 80 | 60.6 | 400 | 1/10–1/160 | 0.9753 | 105.3 |
| AH-F1/AH-qPCR-R2 | 86 | 60.6 | 400 | 1/20–1/160 | 0.9924 | 101.0 | |
| AH-F2/AH-qPCR-R1 | 74 | 60.6 | 400 | 1/20–1/160 | 0.9726 | 102.8 | |
| AH-F2/AH-qPCR-R1 | 80 | 60.6 | 400 | 1/20–1/160 | 0.9993 | 101.3 |
aThe best primer pair for each gene is in bold. bTm, annealing temperature. cE (%), efficiency