Measuring mRNA and miRNA Expression

Leaves (two-week old rosettes), floral clusters (five weeks) and bent cotyledon embryos (eight DAP) were homogenized in 500 μl TRIzol reagent (Invitrogen) and total RNA was isolated and purified according to manufacturer’s recommendations. For mRNA, 200 ng of total RNA was used for cDNA synthesis with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). The cDNA was diluted two-fold for embryos or ten-fold for leaves and floral buds with nuclease-free water. Two μL of diluted cDNA was used as a template for the qRT-PCR with Fast SYBR Green Master Mix (Roche) on a LightCycler 96 instrument (Roche) with two technical replicates for each biorep. For miRNA823 quantification, corresponding stem-loop primers were added to the RT reaction (adapted from Yang et al., 2014 (link)) and miR823 levels were measured using Fast SYBR Green Master Mix (Roche) with miRNA823-specific forward primer and a stem-loop specific universal reverse primer. U6 snRNA was used as the reference RNA (adapted from Shen et al., 2010 (link)). Primers used for qRT-PCR are listed in Supplementary file 5.