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.