Thermal cycle dice real time pcr system

Total RNA was extracted using the Pure Link^TM RNA Mini Kit (Invitrogen, California, CA, USA), according to the manufacturer’s instructions [8 (link),9 (link)]. The quantification of total RNA was performed by a Nano-MD UV-Vis spectrophotometer (Scinco, Seoul, Korea). Complementary DNA (cDNA) was synthesized using RevertAid Reverse transcriptase (Thermo Scientific, Waltham, MA, USA) in a 20 μL reaction volume. Quantitative reverse transcription (qRT) polymerase chain reaction (PCR) was performed and analyzed using TB Green™ Premix Ex Taq™ (Takara, Shiga, Japan) and Thermal Cycle Dice real-time PCR system (Takara, Shiga, Japan) as reported previously [9 (link)]. Three independent replicates were performed for each primer sets. The primer sequences used in this study are provided in Table S1.

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.

Total RNA was extracted from different organs of Arabidopsis using Pure link™ RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) with on-column DNase I treatment to remove residual genomic DNA. Extracted total RNA was verified for quality and quantity using a Nano-MD UV-Vis spectrophotometer (Scinco, Seoul, Korea). The complementary DNA (cDNA) was synthesized in a total 20 μL reaction volume using RevertAid Reverse transcriptase (Thermo, Waltham, MA, USA). qRT-PCR was performed using TB Green™ Premix Ex Taq™ (Takara, Shiga, Japan) and Thermal Cycle Dice real-time PCR system (Takara, Shiga, Japan), as previously reported [10 (link)]. Target gene-specific primers for the qRT-PCR analysis are listed in Table S1.