Miscript sybr premix green pcr kit

Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and was reverse transcribed to cDNA to evaluate the mRNA expression levels of ZNRF3, c-myc, cyclin D1 and TCF4 using the PrimeScript RT Master mix (Takara Bio, Inc.) according to the manufacturer's instructions. The protocol of RT was 37°C for 15 min, 85°C for 5 sec and held at 4°C. The PCR protocol was: Initital denaturation at 95°C for 5 sec, followed by 40 cycles at 60°C for 30 sec and by annealing and extension at 50°C for 30 sec. To evaluate miR-301a, the protocol of miRNA reverse transcription was 37°C for 60 min, 95°C for 5 min and held at 4°C, and stem-loop RT-PCR was performed with an RNA PCR kit (Qiagen GmbH) according to the manufacturer's instructions. qPCR was performed using the miScript SYBR Premix Green PCR kit (Qiagen GmbH) and the Roche LC480 quantitative Real-Time PCR system (Roche Diagnostics). The amplification conditions were: Initital denaturation at 95°C for 15 min, followed by 40 cycles at 94°C for 15 sec and by annealing and extension at 55°C for 30 sec. GAPDH or U6 levels were selected as internal controls, and fold changes were calculated using the relative quantification (2^-ΔΔCq) method (20 (link)). The primer sequences are presented in Table SI. Data were analyzed from three independent experiments and are presented as the mean ± SD.