Sybr green mix

For abiotic stress treatments, 7-day-old Arabidopsis seedlings were exposed to 150 mM NaCl, 300 mM mannitol, 5 mM H₂O₂, 100 µM abscisic acid (ABA), cold (4 °C), or heat (37 °C). The seedlings were collected at different time points (0, 3, 6, 12, and 24 h) after treatment, and then frozen immediately in liquid nitrogen for RNA extraction.
Total RNA was extracted using the RNAprep pure plant kit (Tiangen, Beijing, China), and cDNA was synthesized from 1 µg of total RNA using the M-MLV RTase cDNA synthesis kit (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. qPCR was performed on an Mx3000P QPCR system (Agilent Technologies, Palo Alto, CA, USA). The reaction components per 20 µL were as follows: 10 µL SYBR Green Mix (Agilent), 1 µL 10 µM of each primer and 1 µL cDNA, and 7 µL H₂O. The thermal cycling program was as follows: initial denaturation at 95 °C for 120 s, and 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The Arabidopsis AtActin2 gene was used as an internal control [14 (link)]. The relative quantification of gene expression was evaluated using the delta-delta-Ct method. The transcript level in untreated seedlings (control) was set as 1.0. The primers used in this study are shown in Supplementary Table S1.

Briefly, the total RNA was isolated using the Trizol reagent (Invitrogen, United States) according to the manufacturer’s instructions and then 1 μg RNA was reverse transcribed into cDNA using a reverse transcription kit (Roche) according to the manufacturer’s instructions. Primer pairs used for real-time PCR were shown in Table 1. Real-time PCR (qPCR) experiment was performed using a reaction system containing SYBR Green Mix (Agilent Technologies, Santa Clara, United States), cDNA, and primers. The annealing temperature was 62°C and the cycle of gene magnification was 39. Relative expression of target genes was calculated using the 2^−ΔΔCt method and normalized to GAPDH.

Tissue samples stored in RNAlater (Thermo Fisher Scientific) were processed for RNA extraction using a TissueLyser II and QIAzol reagent (Qiagen). Isolated RNA was quantified using a Qubit fluorimeter and RNA BR kit (Qiagen). cDNA was synthesized using Tetro reverse transcription kit (Bioline) and oligo dT 15-mers (Integrated DNA Technologies). Quantitative real-time PCR was performed using SYBR Green mix (Agilent Technologies) and a LightCycler 480 II (Roche). A list of primer sequences used is shown in Table II.

Total RNA was extracted using a RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 µg total RNA with the M-MLV RTase cDNA synthesis kit (TaKaRa, Shiga, Japan). Real-time quantitative PCR analysis was performed using the SYBR green mix (Agilent Technologies, Palo Alto, CA, USA) in an optical 96-well plate on a Mx3000P system (Agilent). Three biological repeats and three technical repeats were performed for qPCR analysis. The primers used in this study are shown in Supplementary Table S1.

Total RNA was extracted using an RNeasy^® Mini Kit (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized using an M-MLV RTase cDNA Synthesis Kit (TaKaRa, Shiga, Japan). Real-time quantitative PCR (qPCR) analysis was performed using SYBR^® Green Mix (Agilent Technologies, Palo Alto, CA, USA) on an Mx3000P system (Agilent Technologies). Three biological replicates and three technical replicates were performed for each analysis. The primers used in this study are shown in Table 1.

Total RNA was extracted using an RNeasy^® Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 µg of total RNA with the M-MLV RTase cDNA Synthesis Kit (TaKaRa, Shiga, Japan). Real-time quantitative PCR (qPCR) analysis was performed using SYBR^® Green Mix (Agilent Technologies, Palo Alto, CA, USA) in an optical 96-well plate on an Mx3000P system (Agilent Technologies). Three biological replicates and three technical replicates were performed for each analysis. The primers used in this study are shown in Table 1.