Thermo scientific maxima sybr green rox qpcr master mix kit

Expression rate of phenylalanine ammonia-lyase (FcPAL), chalcone synthase (FcCHS) and UDP glucose-flavonoid 3-O-glcosyl-transferase (FcUFGT) genes in start, middle and end point of flavonoids biosynthetic pathway and FcMYB3 gene as transcription factor in HRs of F. carica (Fig. 1) were determined to investigate the impact of elicitation. Details of specific primer sets adopted from Wang et al. (2017) (link) are given in Table 1. Prior to RT-PCR, the RT-PCR products of control HRs cDNA were checked by 1.2% agarose gel electrophoresis to confirm amplicon size of specific primer sets. Each three-step real-time RT-PCR reaction was performed on Rotor-Gene Q cycler (QIAGEN, USA) using Thermo Scientific Maxima ®SYBR-Green/ROX qPCR Master mix kit (Thermo Fisher Scientific, Lithuania) at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing (acquiring on green) at 54-60 °C for 40 s and extension at 72 °C for 45 s. The relative quantitative analysis by 2 -ΔΔCT method was used to determine the fold change in expression level of genes. Actin as an endogenous housekeeping gene (FcACT1) (Ikegami et al., 2013) (link) was used for normalization of real-time PCR data.