Quantitative RT-PCR Analysis of Tobacco Transcripts

Total RNA was extracted from WT tobacco tissue, such as leaves, roots, stems, buds and flowers, and from transgenic tobacco leaves according to the method described in Chen et al. (2017) (link). First-strand cDNA was synthesized using a PrimeScript^TM RT Master Mix (Perfect Real Time) (Takara, Clontech, Japan) according to the manufacturer’s instructions. The qRT-PCR was performed using SYBR^® Premix Ex Taq^TM (Tli RNaseH Plus) (Takara, Clontech, Japan), and primers listed in Supplementary Table S1. The Tob103 gene (GenBank accession no. U60495) served as an internal control. The reaction mixtures consisted in 10 μl SYBR Green mix, 0.4 μl forward qRT-PCR primer, 0.4 μl reverse primer, 1 μl template cDNA, and 7.8 μl sterile water. The thermal cycling parameters were 95°C for 30 s, followed by 40 cycles of 95°C for 15 s and 58°C for 34 s. The relative expression levels were normalized to the expression of the Tob103 gene. The comparative cycle threshold (ΔΔCT) method was used to calculate the relative expression levels of the target genes. Data were expressed as the mean ± SD as determined from three independent biological replicates.