Mx4000 quantitative pcr system

The TRIzol one-step method (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from KGN cells. Total RNA concentration was detected using a nucleic acid protein analyzer (Beckman Coulter, Inc.). RT was immediately performed using the Prime Script RT-PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions to avoid RNA degradation. qPCR analysis was performed using the quantitative SYBR-Green PCR kit (Qiagen GmbH) and the Mx4000 quantitative PCR system (Stratagene; Agilent Technologies, Inc.). The reaction conditions used for the qPCR were as follows: Initial denaturation for 5 min at 95˚C; followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 30 sec and extension at 72˚C for 34 sec. The internal controls used were GAPDH or U6. Gene expression was analyzed using the 2^-ΔΔCq method (17 (link)). The primer sequences for PCR were listed as follows: GAPDH forward, 5'-CTTTGGTATCGTGGAAGGACTC-3' and reverse, 5'-GTAGAGGCAGGGATGATGTTCT-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3' and reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3'; miR-451a forward, 5'-ACACTCCAGCTGGGAAACCGTTACCATTAC-3' and reverse, 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTTACAG-3; ATF2 forward, 5'-TACAAGTGGTCGTCGG-3' and reverse, 5'-CGGTTACAGGGCAATC-3'; and cyclin D1 forward, 5'-CCGTCCATGCGGAAGATC-3 and reverse, 5'-GAAGACCTCCTCCTCGCACT-3'.