Quantitative Gene Expression Analysis

One hundred milligrams of frozen roots of three independent transgenic lines were ground in a Retsch MM300 mixer, and total RNA was extracted using the Qiagen RNeasy kit (Qiagen). One microgram of RNA was used for cDNA synthesis using the iScript^TM cDNA Synthesis Kit (Bio-Rad). qRT-PCR was performed in the LightCycler 480 System (Roche) using the Fast Start SYBR Green I PCR mix (Roche) via the following program: pre-incubation (95°C, 10 s), 45 amplification cycles (incubation 95°C, 10 s; annealing 65°C, 15 s; elongation 72°C, 15 s). Relative expression levels using multiple reference genes were calculated using qBase (Hellemans et al., 2007 (link)). The M. truncatula 40S ribosomal protein S8 and translation elongation factor 1a were used as reference genes. Primer sequences are presented in Supplementary Table 1.