Quantitative Assessment of Gene and miRNA Expression

The isolation of total RNA and synthesis of first-strand cDNA were performed following our previous study [31 ]. To check gene and MIR156b expression, qRT–PCR analysis was performed using a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) according to the protocol recommended by Applied Biosystems using SYBR Premix Ex Taq (Takara Bio, Beijing, China). VvACTIN [62 (link)] and VvUBI [63 ] served as internal controls. AtACTIN was used as a reference control for semi-qRT–PCR in Arabidopsis [64 (link)].
cDNAs were synthesized using an miR156 stem-loop primer and SuperScript III RT–PCR technology to assess the expression of mature miR156b using stem-loop qRT–PCR. As previously stated, a specific reverse transcription primer for mature miRNAs with a stem-loop structure was created [65 (link)]. qRT–PCR reactions (20 μl containing 10 ng cDNA and the vv-miR156b/stem-loop universal-R primer set) were processed using the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), as described above. Here, 5.8S rRNA and U6 were used as the reference [66 (link)]. Each reaction was conducted in three independent biological replicates and verified using melting curve analysis. The relative expressions were calculated using the 2^−ΔΔCT method. Primers used are listed in Supplementary Data Table S1.