2.5. Single tube duplex RT-PCR
The single tube duplex RT-PCR was conducted by the following method. 5μL of 2 × Ace Taq Master Mix, 0.4 μL of MYCN and NAGK forward primer (10 μM), 0.4 μL of MYCN and NAGK reverse primer (10 μM), 0.5 μL of MYCN and NAGK MB (10 μM), 1μL of synthetic target DNA or total DNA extracted from real samples and 2.7 μL DEPC water were mixed to a final volume of 10 μL. The RT-PCR was conducted under the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 10 s, 28 °C for 30 s and 60 °C for 30 s. The fluorescence signal was detected at 28 °C.