Quantitative reverse transcription-PCR

Gene transcription levels were measured using the qRT-PCR method as described previously [24]. Total RNA was extracted using TRIzol LS Reagent (Invitrogen) and the concentration was measured. Quantitative PCR was performed in triplicate using SensiFAST SYBR Hi-ROX One-Step Kit according to the manufacturer's instructions (Bio-73005, Bioline). The mRNA level for each gene was detected by Applied Biosystems™ Real-Time PCR Instruments. Primers used in this study were MYCN: Forward 5’-AGAGGACACCCTGAGCGATTC-3’, Reverse 5’-CATAGTTGTGCTGCTGGTGGA-3’; MYC: Forward 5’-CTCCATGAGGAGACACCGCCCA-3’, Reverse 5’-AAGGTGATCCAGACTCTGACCT-3’; MELK: Forward 5’-ATAGCTACCATCTCTCCAGTA-3’ and Reverse 5’-CTTGCAAGAGGACTATGAAAG-3’, respectively.