Gene Expression Analysis by qPCR

Total RNA extraction was performed using Pure link^TM RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the RNA quality was vitalized by agarose gel loading (Figure S3). The extracted total RNA was quantified using a spectrophotometer (Nano-MD UV-Vis, Scinco, Seoul, Korea). RevertAid Reverse transcriptase (Thermo, Waltham, MA, USA) was used in a 20 μL reaction volume to synthesize the complementary DNA (cDNA). Real-time quantitative PCR (qPCR) was performed using TB Green™ Premix Ex Taq™ (Takara, Shiga, Japan) and Thermal Cycle Dice real-time PCR system (Takara, Shiga, Japan) as previously described [4 (link)]. To determine the relative fold-differences in template abundance for each sample, the Ct value for each of the analyzed genes was normalized to the Ct value for β-actin (At5g09810) and was calculated relative to a calibrator using the formula 2-ΔΔCt. Three independent experiments were performed for each primer set (Table S1). The primer efficiency was determined according to the method of Livak and Schmittgen [25 (link)] in order to validate the ΔΔCt method. The gene-specific primer sequences for the target genes are listed in Supplementary Table S1.