Thunderbird sybr green master mix

Reverse transcription of the extracted total RNA (1 μg) was performed using a 5 × Master Mix (TOYOBO, Osaka, Japan). The reaction mixture was prepared according to the manufacturer’s instructions. Quantitative real-time PCR was carried out on each cDNA sample using THUNDERBIRD SYBR Green Master Mix (TOYOBO, Osaka, Japan) and an ABI Step One Real-Time PCR System (Applied Biosystems, Foster City, CA, USA)(Mekawy et al. 2018 (link)). OsUBC was used as an endogenous control gene in the roots. The reaction mixture contained 7.5 µL SYBR Mix, 1.5 µL forward primer, 1.5 µL reverse primer, 0.3 µL 50 × ROX reference dye, 1 µL cDNA, and 3.2 µL milli-Q H₂O. The following cycling parameters were used: initial incubation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 15 s and extension at 60 °C for 60 s, and melting curve analysis. The relative gene expression levels were calculated using the comparative 2^−ΔΔCT method. Data represent the average of three technical replicates.

Gene expression levels were examined by quantitative PCR analyses. Briefly, total RNA was isolated from cells using ISOGEN II (Nippon Gene, #311-07361) and purified using RNeasy Mini Kit (Qiagen). Total RNA (500 ng) was reverse-transcribed to cDNA using ReverTra Ace (Toyobo, #FSQ-101). Quantitative PCR was performed using Thunderbird SYBR Green master mix (Toyobo, #QPS-201) and a StepOne Plus Real-Time PCR System (Thermo Fisher). Primers used for the quantitative PCR analyses are listed in Table S2.