RT-qPCR Analysis of Tobacco Leaf and Stem Genes

Total RNA was isolated from 2-month-old wt and F₃1 leaves and stems as previously reported [29 (link)]. RT-qPCR analysis was performed using SYBR Green fluorescent detection in a CFX96 Real-Time System Cycler (Bio-Rad, Hercules, CA, USA) with three biological and three technical replicates per sample. The primer sequences used are reported in Table S1. The PCR program was as follows: 10 min at 95 °C; 50 cycles of 15 s at 95 °C; 20 s at 60 °C; and an increment of 0.5 °C every 0.5 s from 65 °C to 95 °C. The specificity of PCR products was checked in a melting-curve test. The Nicotiana tabacum Elongation factor EF-1α (EF-1α, AF120093), L25 ribosomal protein (L25, L18908), and Ubiquitin-conjugating enzyme E2 (Ntubc2, AB026056) genes were tested as reference genes [30 (link)]. In wt and F₃1 samples, all genes had a variation coefficient below 0.1, according to Czechowski et al. [31 (link)]. The EF-1α gene was chosen as the reference gene because it showed the lowest variation coefficient among the genes (Table S2). Differences in gene expression between transformed and wt samples were considered significant when the expression was at least doubled (greater than or equal to two-fold upregulation) or halved (less than or equal to two-fold downregulation), according to Chen et al. [32 (link)].