Arabidopsis RNA Extraction and qRT-PCR

Total RNA was extracted from roots of 10-day-old Arabidopsis WT (Col-0) with TRIZOL reagent (Life Technologies, Mulgrave, VIC, Australia) according to Chen et al. (2015b ) and Liu et al. (2014) (link). RNA was reverse transcribed with a sensiFAST Kit (Bioline, Alexandria, NSW, Australia) and real-time PCR was performed with a SensiMix SYBR No-ROX Kit (Bioline, Alexandria, NSW, Australia) using a Rotor-Gene Q6000 (QIAGEN, Hilden, Germany). The gene-specific primers are as below: forward 5'-CACTATGGCAACTGTCGGTTATGG-3' and reverse 5'-GCGGTTCATGAAACTTATGAGATC-3' for GORK; and forward 5'-TGCGGGTGCCCATTTAAC-3' and reverse 5'-CTTTCACAAACCACCAGTAGC-3' for RBOHD. Amplification of the RNA polymerase II subunit (RPB2) (forward primer 5'-TTCCCCGTTCCGATAACT-3' and reverse primer 5'-ATGCTCTGCCGTCCACC-3') gene was used as an internal control. The PCR program was two steps: one cycle of 95 °C, 10 min; 40 cycles of 95 °C, 15 s; 60 °C, 15 s; 72 °C, 15 s. The amplification of the target genes was monitored every cycle by SYBR-green fluorescence. Three biological and three technical replicates were performed for each treatment.