Fast start sybr green 1 pcr mix

Frozen plant material was ground in a Retsch MM300 mixer and total RNA was extracted using the Qiagen RNeasy kit (Qiagen, http://www.qiagen.com/). An RNase-free DNase step was performed following manufacturer's instructions for preparation of RNA. Next, 1 µg of total RNA was used for cDNA synthesis with the iScript kit (Bio-Rad, http://www.bio-rad.com/). RT-PCR was performed on a LightCycler 480 system (Roche, http://www.roche.com) using the Fast Start SYBR Green I PCR mix (Roche). The primer sequences are provided as Table S2. For TIFY8 primer pair #4 was used unless stated otherwise. Samples were amplified as described: one pre-incubation step (95°C, 10 s) followed by 45 amplification cycles (incubation 95°C for 10 s, annealing at 65°C for 15 s, elongation at 72°C for 15 s). Primer efficiency was at least 1.7. Gene expression levels were quantified relative to the housekeeping gene UBC (AT5G25760).

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.

Seedlings were grown in the same conditions as in Iñigo et al. (2016) (link). Seedlings were frozen in liquid nitrogen and total RNA was extracted using RNeasy plant mini kit (Qiagen) and DNAse I (Promega) treatment. Next, 1 µg of RNA was used for cDNA synthesis using iScript kit (Bio-Rad). qRT-PCR was performed on a LightCycler 480 system (Roche) using the Fast Start SYBR Green I PCR mix (Roche) with three biological repeats and three technical repeats. Data were analyzed using the second derivative maximum method and relative expression levels were determined using the comparative cycle threshold method.