RNA isolation, and primers and qPCR
RNA was extracted with TRIzol™ Reagent (Invitrogen, Germany) following the guideline provided by the manufacturer. Traces of DNA in the RNA samples were digested with TURBO™ Dnase (Thermo Fisher Scientific, Germany), and cDNA synthesis was performed with Omniscript RT Kit (Qiagen, Germany), following manufacturer’s instructions.
Each 20 μL qPCR reaction contained 2 μL of 10 x DreamTaq Buffer (Thermo Fisher Scientific, Germany), 0.2 mM dNTP, 0.5 μM forward/reverse primer, 40 ng cDNA, 1 μL 20 x Evagreen® (Biotum, Germany) and 1.5 U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Germany). Real-time PCR reaction was conducted with CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, Germany). The initial denaturation step was set at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 50 s, and extension at 72 °C for 1 min. Melt curve analysis was performed by incubating at 95 °C for 10 s, 65 °C 5s, and increase to 95 °C at 0.5 °C/5 s increment. Melt curve analysis showed a single peak for all genes analyzed. Values were normalized to the housekeeping gene RPS18B (AT1G34030) for gene expression analysis. Gene-specific primer pairs used in this study and the gene accession numbers are listed in Table S1.