miRNA Expression Profiling via RT-qPCR

The cDNA of mature miRNA was prepared using miRNA reverse transcription kit M-MLV (Takara, China), and the reverse-transcribed products were used as template for RT-qPCR with gene-specific primers. The miRNA specific stem-loop primers and gene-specific RT-qPCR primers were designed according to the protocol described previously (Chen et al., 2005 (link)). For target gene validation, the cleavage site-spanning fragments of GRF genes were detected. Total RNA extracted from different tissue types was reverse-transcribed by oligo (dT) primed cDNA synthesis protocol (Fermentas). The resulting cDNA was subjected to quantitative PCR using Bio-Rad CFX-96 RealTime PCR System (Bio-Rad, United States) in a final volume of 20 μl containing 2 μl of cDNA and 10 μl of SYBR premix Ex-taq™ (Takara, Japan). Ubiquitin was used as an internal control for normalization to compare gene expression level between the accessions. For each reported result, at least three independent biological samples were subjected to a minimum of three technical replicates. The primers designed with Primer 7.0 software are listed in Supplementary Table 1.