Arabidopsis Root Total RNA Extraction and RT-PCR

Total RNA was extracted from 10-day-old Arabidopsis roots with TRIZOL reagent (Life Technologies, Mulgrave, VIC, Australia) (Liu et al., 2014 (link); Chen et al., 2016 (link)). First-strand cDNA synthesis was performed using the sensiFAST Kit (Bioline, Alexandria, NSW, Australia) with 1 µg of total RNA. RT-PCR was performed with SensiMix SYBR No-ROX Kit (Bioline, Alexandria, NSW, Australia) using a Rotor-Gene Q6000 (QIAGEN, Hilden, Germany). The specific primers are listed in Supplementary Table S1 at JXB online. The PCR program was two steps: one cycle of 95 °C, 10min; and 40 cycles of 95 °C, 15s; 60 °C, 15s; and 72 °C, 15s. The amplification of the target genes was monitored every cycle by SYBR-green fluorescence. Three technical and biological replicates were performed for each experiment and treatment. The transcripts of target genes were normalized to the control gene RNA polymerase II subunit (RPB2).