Quantifying TgNAC01 Gene Expression

Total RNA was extracted from leaves of teak and tobacco transgenic lines using the TRIzol reagent (Thermo Fisher, Waltham, MA, USA) to examine the TgNAC01 gene expression. Total RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and its integrity was examined by electrophoresis. The RNA was treated with DNAse I (Promega, USA) and cDNA was synthesized using the SuperScript™ III First-Strand Synthesis System for RT-qPCR (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) reactions were carried out using a Platinum Sybr Green Supermix (Invitrogen, Waltham, MA, USA) and run in an ABI 7500 qPCR thermocycler (Applied Biosystems, Waltham, MA, USA). Expression data were normalized using the 2-ΔCt method. The constitutive Elongation Factor-1 alpha (EF-1 alpha) housekeeping gene was used as internal control for tobacco (Schmidt & Delaney, 2010 (link)) and teak (Galeano et al., 2014 (link)). Primers were designed using the primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/) (Table S1). We used three biological replicates and two technical repetitions for each transgenic line.