Quantifying miRNA Expressions using RT-qPCR

The mature miRNA reverse transcription was performed with miR-specific stem-loop forward primers and a universal reverse primer, URP. These miR-specific primers were designed according to the mature miRNA sequence [73 (link)] (Additional file 13). For real-time PCR, cDNA was mixed with 2 × SYBR Green Mix (Takara, Japan) and each of the miRNA specific primers and universal reverse primer in a final volume of 20 μl. The PCR process was performed using SYBR Green as fluorescence dye and run on 96-wells plates with the ABI 7500 FAST Real Time system (Applied Biosystems). PCR was performed with 95 °C for 30 s, 40 cycles of 95 °C for 3 s, 60 °C for 30 s and 72 °C for 30 s. Ubiquitin 7 (GhUBQ7) (Additional file 13) was used as the reference gene [74 (link)]. The relative expression level of miRNA was calculated by 2^-△△Ct. Three biological replicates were performed.