Sybr realmastermix

Stem-loop qRT-PCR was carried out to validate the presence and expression of the identified miRNAs. Total RNA was extracted from leaves using Trizol (TaKaRa, Dalian, China), according to the manufacturer’s instructions, and the RNA concentrations and integrity were analyzed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis. Finally, the RNA was treated with RNase-free DNase I (TaKaRa) to remove genomic DNA. Sequence-specific forward primers were designed for 9 selected miRNAs (S1 Table), and qRT-PCR was performed using SYBR RealMasterMix (Tiangen Biotech, Beijing, China) and the ABI 7300 real-time PCR detection system (Applied Biosystems, USA), with the following amplification conditions: denaturation at 95°C for 3 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 61°C for 30 s. Finally, melting analyses were performed to confirm the absence of false-positive peaks. All reactions were performed in triplicate for each sample, and U6 snRNA was used as an internal reference [28 (link)]. The relative expression levels of the miRNAs were calculated using the 2^-ΔΔCt method [29 (link)].