Table 1.
Major differences in RT-qPCR conditions in the two versions of TILDA.
| Characteristic | TILDA | TILDA v2.0 |
|---|---|---|
| Input volume of cells per reaction 1 | 1 µL | 10 µL |
| One-step RT-PCR reagents | Superscript III Platinum Taq (Life Technologies) & Buffer, RNase inhibitor (Life Technologies), Tris-EDTA (TE) buffer | HotStarTaq DNA Polymerase, Sensiscript and Omniscript Reverse Transcriptases & Buffer (QIAGEN), RNasin Ribonuclease Inhibitor (Promega), 0.1% solution of Triton X-100 |
| Final reaction volume | 11 µL | 50 µL |
| Cycling conditions | 50 °C for 15 min, 95 °C for 2 min, 24 cycles of amplification (95 °C 15 s, 60 °C 4 min) | 50 °C for 30 min, 95 °C for 15 min, 25 cycles of amplification (95 °C 30 s, 55 °C 1 min, 72 °C 2 min) final extension at 72 °C for 5 min |
| Input volume of pre-amplified product per reaction | 1 µL of 1:5 dilution | 2 µL |
| Real-time PCR reagent | LightCycler 480 Probe Master buffer (Roche Applied Sciences) | Custom TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) |
| Final reaction volume | 10 µL | 20 µL |
| Cycling conditions | 95 °C for 10 min, 45 cycles of 95 °C 10 s, 60 °C 30 s, 72 °C 1 s and a cooling step at 40 °C for 30 s | 50 °C for 5 min (UNG step), 95 °C for 20 s, 45 cycles of 95 °C for 3 s and 60 °C for 30 s |
1 Cells are serially diluted and then distributed in replicates of 18,000, 9000, 3000 and 1000 per well with the indicated volume. Primers and probe as published [17].