Sybr green maxima sybr green rox qpcr master mix 2x

Pooled total RNA (1.0 μg) from 5 plants, from two independent experiments, was retro-transcribed into cDNA according to the manufacturer's indications using the SCRIPT cDNA Synthesis Kit (Jena Bioscience www.jenabioscience.com). RT-qPCR was performed in 96-well plates with the Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific), using SYBR Green Maxima SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, www.thermofisher.com). Two independent experiments were analyzed with three technical replicates each. RT-qPCR conditions were as follows: an initial 95°C denaturation step for 15 min followed by denaturation for 15 s at 95°C, annealing for 30 s at 60°C, and extension for 30 s at 72°C for 45 cycles. Gene expression values were normalized using the mean expression of two genes: AT4G26410 and AT1G72150 previously described as stable reference genes (Serrano and Guzmán, 2004 (link); Czechowski et al., 2005 (link)). Normalized gene expression was determined using the comparative 2^−ΔΔCT method previously described (Schmittgen and Livak, 2008 (link)). Primers for ACS6, PR4, and PDF1.2 gene expression were previously described (Hael-Conrad et al., 2015 (link)).

The pooled total RNA (1.0 µg) used for RNAseq analysis was retro-transcribed into cDNA according to the manufacturer’s indications using the SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). qRT-PCR was performed in 96-well plates with the Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA) using the SYBR Green Maxima SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, Waltham, MA, USA). Three independent experiments were analyzed, each with three technical replicates. The qRT-PCR conditions were as follows: an initial 95 °C denaturation step for 5 min, followed by denaturation for 15 s at 95 °C, annealing for 30 s at 60 °C, and extension for 30 s at 72 °C for 45 cycles. Gene expression values were normalized using the mean expression of two genes: AT4G26410 (RHIP1) and AT1G72150 (PATL1), which were previously described as stable reference genes [44 (link),78 (link)]. Normalized gene expression was determined using the comparative 2^−∆∆CT method, as previously described [79 (link)]. Primers for gene expression analysis have been previously described: for PR1, PDF1.2 and PR4 [43 (link)]; for ZAT12 [37 (link)], ICS1 [80 (link)] and ACS6 [81 (link)].