Mircute plant mirna isolation kit

Total RNA was extracted from cotton using the miRcute Plant miRNA Isolation Kit (Beijing Tiangen Biotech, Beijing, China). RNA integrity was assessed by 1.2% agarose gel electrophoresis with 1 × TAE electrophoresis buffer at 130 V for 20 min. RNA with a 28S:18S ratio of 2:1 was used for subsequent experiments. The concentration of total RNA was measured by a Nanodrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA).
The cDNA of the target genes was synthesized using the Easyscript^® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing TransGen Biotech, Beijing, China). In a 20 µL reaction system, 1 µg total RNA, 10 µL 2 × ES reaction mixture, 1 µL Oligo(dT)₁₈ primer, 1 µL Easyscript^® RT/RI Enzy Mix, 1 µL gDNA remover, and variable RNase-free water were mixed. The mixture was incubated at 42 °C for 30 min, followed by 85 °C for 5 s. For miRNA cDNA synthesis, a miRNA-specific stem-loop RT primer was used. The reaction was incubated at 16 °C for 30 min, followed by 30 °C for 30 s, 42 °C for 30 s, and 50 °C for 20 s for 60 cycles. Finally, the reaction was stopped at 85 °C for 5 s.

miRNAs were isolated from the petals of 42 tree peony varieties using the miRcute Plant miRNA Isolation Kit (Tiangen, Beijing, China). Then, miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen, China) was used to synthesize the first strand. Reaction system (20 μL): Total miRNA 2 μL, 2× miRNA RT Reaction Buffer 10 μL, miRNA RT Enzyme Mix 2 μL, and RNase-free ddH₂O 6 μL. The reaction procedure was as follows: 42 °C for 60 min and 95 °C for 3 min.