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.