Depc h2o

The mixture contained 2 nM each of template miRNA and probes A and B. The procedure was initially performed at 65°C for 8 min. In this step, both the DNA probes identify and hybridize with the target miRNA. Then, 2 U of T4 RNA ligase II and 10× ligation buffer (New England Biolabs, Shanghai, China) were added to the hybridization product, and a total final volume of 50 μl was incubated at 37°C for 1 h to perform the ligation reaction; the products were then immediately placed on ice until it cooled to room temperature (∼15 min).
The FastStart Universal SYBR^® Green Master (Roche, Mannheim, Germany) was used for real-time quantitative PCR assays. The quantitative PCR assays for amplifying the target miRNA were performed with a 20-μl final volume containing 2× SYBR Green Mastermix (ROX), 0.2-μM forward and reverse primers, 5 μl of ligation production, and 4.2 μl of diethylpyrocarbonate-treated water (DEPC-H₂O; Takara Biotechnology, Changchun, China). The reactions were incubated at 95°C for 5 min on a Bio-Rad CFX96 Real-time Thermal Cycler (Bio-Rad, Hercules, CA, United States), followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and then 72°C for 30 s. All reactions were performed in triplicate.

RNase H (TaKaRa) was added to the obtained reverse-transcribed products to degrade free RNA. To synthesize double-strand cDNA (dscDNA), anchored random primers were added and incubated at 65°C for 5 min and then placed on ice for 5 min for denaturation (Hameed et al., 2020 (link)). After this, 1 μL of Klenow fragment (TaKaRa), 1 μL of 10 mM dNTPs (TaKaRa), 2 μL of 10 × Klenow buffer (TaKaRa), and 6 μL of ddH₂O (TaKaRa) were added and incubated at 37°C for 60 min, followed by an incubation at 75°C for 10 min. To remove phosphates and the free single-strand nucleic acid in the dscDNA reaction, 0.5 μL of Exonuclease I (TaKaRa), 1 μL of alkaline phosphatase (TaKaRa), 5 μL of 10 × phosphatase buffer (TaKaRa), and 24 μL of DEPC H₂O (TaKaRa) were added and incubated at 37°C for 60 min, followed by an incubation at 75°C for 10 min.

Before the synthesis of dscDNA, 1 μL of RNase H (TaKaRa) was added to the obtained products to degrade the free RNA. To synthesize dscDNA, anchored random primers were added and incubated 75°C for 5 min and then on ice for 5 min for denaturation. Then, 1 μL of Klenow fragment (TaKaRa), 1 μL of 10 mM dNTPs (TaKaRa), 2 μL of 10 × Klenow buffer (TaKaRa), and 6 μL of dd H₂O (TaKaRa) were added and the samples were incubated at 37°C for 60 min, followed by 75°C for 10 min. Then, 0.5 μL of exonuclease I (TaKaRa), 1 μL of shrimp alkaline phosphatase (SAP, TaKaRa, Dalian, China), 5 μL of 10 × phosphatase buffer (TaKaRa), and 24 μL of DEPC H₂O (TaKaRa) were added and incubated at 37°C for 60 min followed by 75°C for 10 min to eliminate the phosphates and the free single-strand nucleic acid in the dscDNA reaction.