Maxima sybr green qpcr master mix 2 kit

qPCR experiments were performed in a StepOne™ Real-Time PCR system (Applied Biosystems, Sourceforge, USA) using the Maxima™ SYBR Green qPCR Master Mix (2×) kit (Fermentas, Ontario, Canada), following manufacturer’s instructions. Each reaction contained 2.5 mM MgCl₂ and 2 µM of each primer were used in 25 µL volume reactions, with 4 µL of cDNA as template. A control without cDNA template was included in each set of reactions. Primer sequences are provided in Supplementary Table S5. For all genes, thermal cycling started with a 95 °C denaturation step for 10 minutes followed by 40 cycles of denaturation at 95 °C for 15 seconds and annealing at gene specific temperature (Supplementary Table S5) for 30 seconds. Dissociation curve analysis was performed to confirm single product amplification and the existence of non-specific PCR products (Supplementary Fig. S8). Three biological replicates and two technical replicates were used for each sample. Gene expression (fold change) was calculated as described in^{81 (link)}. Elongation Factor 1-alpha (EF1α) and Ubiquitin-conjugating enzyme (UBQ) coding genes were used for expression data normalization as previously described^{82 (link)}.

The qPCR experiments were performed in a StepOne™ Real-Time PCR system (Applied Biosystems, Sourceforge, USA) using the Maxima™ SYBR Green qPCR Master Mix (2×) kit (Fermentas, Ontario, ON, Canada), according to the manufacturer’s instructions. For all genes, the PCR cycling parameters were thermal cycling started with a 95 °C denaturation step for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/ elongation at gene-specific temperatures (Table S1) for 30 s. Non-template controls were added. To confirm single-product amplification and the existence of non-specific PCR products, a dissociation curve analysis was performed (Figure S1). Gene expression (fold change) was calculated as described in Hellemans et al. [35 (link)]. For expression data normalisation, Elongation Factor 1-alpha (EF1α) and Ubiquitin-conjugating enzyme (UBQ) coding genes were used, as described previously [36 (link),37 (link)].