Oligo dt primed cdna synthesis protocol

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.

Individual floral organs of wild-type and multi-tepal mutant were used, independently, to extract RNA. The cDNA of the mature miRNA was prepared using the miRNA reverse transcription kit M-MLV (Takara, China), and the reverse-transcribed products were used as the template for RT-qPCR with gene-specific primers. The U6 snRNA was used for normalization. The miRNA specific stem-loop primers and gene-specific RT-qPCR primers were designed according to the rules described in Chen et al. [68 ]. For the RT-qPCR of target genes, total RNA extracted from different tissue types were reverse-transcribed by oligo(dT) primed cDNA synthesis protocol (Fermentas). The resulting cDNA was subjected to relative quantitative PCR using Bio-Rad CFX-96 RealTime PCR System (Bio-Rad, USA) 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 make a comparison of 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 5.0 software are listed in Additional file 14.

For the qRT-PCR of target genes, total RNA extracted from different tissue types was reverse-transcribed using a oligo(dT) primed cDNA synthesis protocol (Fermentas). The resulting cDNA was subjected to relative quantitative PCR using Bio-Rad CFX-96 RealTime PCR System (Bio-Rad, USA) in a final volume of 20 μl containing 2 μl of cDNA and 10 μl of SYBR premix Ex-taq™ (Takara, Japan). Ubiquitin and Actin were used as internal controls for normalization to make a comparison of 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 5.0 software are listed in Additional file 1: Table S1. Genbank accession numbers of the MADS-box genes examined are MF474250, MF474239, MF474243, MF462094, MF462081, MF462082, MF462083, MF474256, MF474244, MF474248, and MF462093.