Sybr green premix pcr kit

Primer sets used in RT-PCR and qPCR are shown in Table 1. RT-PCR was performed using Go Taq Green master mix (Promega) according to the manufacturer's protocol. PCR reactions were performed as follows: 28 cycles of denaturation for 45 s at 94°C, annealing for 45 s at 58°C, and extension for 1 min at 72°C. Then the reaction mixture was electrophoresed on 2% agarose gels with ethidium bromide. We performed qPCR using the SYBR Green premix PCR kit (TaKaRa Bio). Each PCR reaction mix contained 50% SYBR Premix Ex Taq, 10 μM of each primer, and 20 ng cDNA template. The qPCR reactions were run on the CFX96 TM Real Time System (Bio-Rad, Hercules, CA, USA). The cycling conditions comprised initial denaturation at 95°C at 1 min, followed by 40 cycles of denaturation for 5 s at 95°C, and annealing and extension for 1 min at 60°C. To ensure specificity, melting curve analyses were performed by slowly raising the temperature of the sample from 60°C to 95°C. Temperature curves showed a single amplified product with complete absence of primer-dimer formation. The Gene study tools software (Bio-Rad) was used to determine a normalizing factor from β-actin as the internal reference gene, which was used to calculate normalized expression for the target genes.