Semi-qPCR and Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) Analysis in Transgenic Brachypodium

To confirm the seed-expression level of AtCYP85A2 in transgenic plants, the roots, shoots (leaves), and seeds of transgenic plants were harvested, and total RNA extraction was performed with TRI Reagent (Invitrogen, Carlsbad, CA, USA). cDNAs were synthesized from 1 μg of the total RNAs by means of an MMLV-reverse transcription system (Promega, Madison, WI, USA) according to the manufacturer's instructions. PCR of BdGAPDH and AtCYP85A2 was performed using cDNA extracted from the same amount of each tissue as the template. BdGAPDH was used as the reference gene in Brachypodium. BdGAPDH and AtCYP85A2 were amplified using 20 thermal cycles (95°C for 10 s, 58°C for 10 s, and 72°C for 10 s).

Total RNA extracted from transgenic Brachypodium was analyzed by qRT-PCR for BR metabolic, signaling and seed size determining genes. Quantitative RT-PCR was performed with the CFX96^TM Real-Time PCR Detection system (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad). The thermal and cycling condition was 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 50°C for 15 s, and 75°C 15 s. Gene expression levels were normalized to BdGAPDH. The primers used for the semi-qPCR and qRT-PCR are described in Supplementary Table 1.