Trizol tri reagent solution

The total RNA was extracted using Trizol TRI Reagent Solution (ThermoScientific) according to the manufacturer’s standard protocol, cDNA was synthesized according to the manufacturer’s instructions from the total RNA using the reverse transcription BioFact^TM RT Kit. The real-time polymerase chain reaction was conducted using an Eco^TM real-time PCR system (Illumina) using 2× Real-Time PCR Master Mix, including SYBR Green I (Biofact) and 10 pmol of each primer, processed in a two-step PCR program. All analyses were conducted in triplicate, and gene expression data were analyzed using ECO Study program (Illumina). Relative expression levels were determined by testing treated and control plants, and normalized to GmActin.

Total RNA was extracted from the leaves collected 4, 8, 16, and 32 h after exposing samples to different treatments. Total RNA was extracted with Trizol TRI Reagent Solution (ThermoScientific, Seoul, Republic of Korea) according to the manufacturer’s instructions. The obtained RNA was subjected to cDNA synthesis and quantitative PCR (qPCR) according to the detailed methods of Kazerooni (2018) [111 (link)] using a reverse transcription kit (BioFactTM RT, Daejeon, Republic of Korea). Two-step real-time polymerase chain reactions were conducted using an EcoTM real-time PCR system (Illumina) and real-time PCR master mix, which included SYBR Green I (Biofact) and 10 pmol of each primer. ECO Study (Illumina) was used to analyze gene expression data in triplicate. Relative expression levels of various genes (TDC, T5H, SNAT, and OMT) involved in the melatonin pathway were determined in control and treated plants (Supplementary Table S1).