Abi 7500 real time pcr system

Total RNA was extracted from cells with TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Then the total RNA was reversed into cDNA in accordance with the instructions of PrimeScript RT Reagent Kit (Beyotime, China), and quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted using the SYBR PrimeScript RT-PCR Kit (TaKaRa) with ABI 7500 Real-Time PCR System (Agilent Technologies, USA). Primer sequences for PCR were listed as following:
GAPDH was used as the internal control. The target gene relative levels were analyzed by the 2^−ΔΔCq method [24 (link)].

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from KGN cells following the manufacturer's protocol. Subsequently, cDNA was synthesized from the total RNA using a cDNA Synthesis kit (Takara Bio, Inc.). RT conditions were as follows: 25˚C for 5 min, 42˚C for 60 min and 80˚C for 2 min. RT-qPCR was conducted using an ABI 7500 Real-Time PCR System (Agilent Technologies, Inc.) with SYBR Green Master mix (Takara Bio, Inc.). The samples were initially incubated at 95˚C for 10 min to denature the cDNA strand and 37 cycles were performed including denaturation at 95˚C for 30 sec, annealing at 60˚C for 60 sec and extension at 72˚C for 15 sec. A final extension was conducted at 72˚C for 10 min. GAPDH was used as the internal reference for mRNA and U6 for miRNA. Primer sequences for PCR were as follows: GAPDH forward, 5'-TTTGGTATCGTGGAAGGACTC-3' and reverse, 5'-GTAGAGGCAGGGATGATGTTCT-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3' and reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3'; Wnt3a forward, 5'-GTTCGGGAGGTTTGGG-3' and reverse, 5'-CCAGGAAAGCGGACCAT-3'; β-catenin forward, 5'-GTTGTACTGCTGGGACCCTT-3' and reverse, 5'-CCCAAGCATTTTCACCAGCG-3'; and miR-451a forward, 5'-ACACTCCAGCTGGGAAACCGTTACCATTACT-3' and reverse, 5'-CTGGTGTCGTGGAGTCGGCAA-3'. The gene expression levels were calculated using the 2^-ΔΔCq method (28 (link)).