Sybr pcr master mix

Total RNA was extracted from D. suzukii or D. melanogaster y 1 w 1118 adult flies subjected to the monoterpene exposures using Aurum Total RNA Mini Kit (Bio-Rad, USA). One µg of RNA was treated with DNase I (Thermo Fisher, USA) and used for cDNA synthesis, carried out with the OneScript ® cDNA Synthesis Kit (Abm, Canada), according to the manufacturer's instructions.
Real time PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) in a 12 µl reaction mixture containing 1.6 µl cDNA (diluted 1:2), 6 µl Sybr PCR Master Mix (Vazyme, China), 0.4 µl forward primer (10 µM), 0.4 µl reverse primer (10 µM) and 3.6 µl nuclease free water. Thermal cycling conditions were: 95 °C for 2 mins, 40 cycles at 95 °C for 15 s and 60 °C for 20 s. After the cycling protocol, a melting-curve analysis from 55 °C to 95 °C was applied. In D. suzukii expression of TAR1 was normalized using AK and TBP genes that served as reference genes (Zhai et al., 2014) . In D. melanogaster y 1 w 1118 expression of TAR1 was normalized using actin and tubulin genes that served as reference genes (Ponton et al., 2011) . Gene-specific primers (Table 1) were used and four independent biological replicates, made in triplicate, were performed for each sample.