Sybr green 1 master kit

To confirm that T-DNA lines are true knockout lines, their RNA was extracted from three-week-old plants rosettes using TRIzol solubilization and extraction [32 (link)] followed by RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands). First strand cDNA synthesis was performed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories N.V., Temse, Belgium) using 1 μg of total RNA used as input material. The 5-fold diluted cDNA and gene-specific primers (Supplementary Table) were used for RT-qPCR performed by iCycler iQ (Bio-Rad Laboratories N.V., Temse, Belgium), with SYBR Green I Master Kit according to manufacturer’s instructions. Data was analyzed by qBASE+ (Biogazelle, Zwijnaarde, Belgium), using ELONGATION FACTOR 1α (EF-1α) and POLYUBIQUITIN 5 (UBIQ5) as reference genes. For each data point three biological and three technical replicates were used.

Analysis of miR expression was performed using the poly(T) adaptor RT-PCR method by Mir-X miR First-Strand Synthesis kit (Clonetech, Takara Bio, Moutain View, CA, USA) as per manufacturer’s instruction. Briefly, for polyadenylation and cDNA synthesis, 1 μg of DNaseI-treated total RNA was incubated at 37 °C for 60 min in a 10 μL reaction volume containing mRQ enzyme, and then the reaction was terminated at 85 °C for 5 min to inactivate the enzymes. Quantitative RT-PCR (qRT-PCR) was run on a TaKaRa Dice Real-Time apparatus (Takara, Shiga, Japan) with the SYBR Green I Master kit (Bio-Rad, Hercules, CA, USA). The reaction conditions for qRT-PCR included the following steps: 10 s at 95 °C followed by 40 cycles of denaturation for 10 s at 95 °C and annealing for 20 s at 60 °C, and extension for 15 s at 72 °C. miRs were quantified using specific primer pairs with the translation initiation factor elongation factor 1-α (EF1α) as the normalization control. Relative transcript abundance was calculated using the 2^−ΔΔCT method [125 (link)]. All experiments were performed using three biological replicates and three technical replicates. The miR169 family primers were adapted from Serivichyaswat et al. [126 (link)]. All the primers used in the study are listed in Supplementary Table S4.