Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was extracted from 40 mg fresh weight of two-week-old seedlings using a Qiagen (Manchester, UK) RNeasy plant mini kit with on-column DNase treatment as per manufacturer’s instructions. RNA quality and quantity were measured using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). cDNA (500 ng) was produced using Thermo Scientific RevertAid kit with oligo (dT)18 primers as per manufacturer’s instructions. Primers for qRT-PCR were designed using Primer3 or NCBI Primer-BLAST. A list of qRT-PCR primers used is provided (Supplemental Table 2). qRT-PCR was performed with SYBR Green PCR kits (Qiagen) which includes premixed HotStarTaq DNA polymerase, SYBR Green I dye and dNTPs. Samples were analysed in a Qiagen Rotor-Gene Q cycler, and melt curve analysis was also performed. Each experiment was performed independently three times providing three biological replicates, and three technical replicates were performed within each experiment.