Biospin mirna extraction kit

TRIzol reagent (Invitrogen, USA) was used to isolate total RNA from tissues and cells. The Biospin miRNA Extraction Kit (Bioer Technology, China) was used to extract miRNAs according to the manufacturer’s instructions. Evo M-MLV RT Premix (Accurate Biology, China) and Mir-X™ miRNA First-Strand Synthesis (Takara, USA) were used to synthesise cDNA from total RNA and miRNA, respectively. qRT-PCR was performed using the Premix Pro Taq HS qPCR Kit (Accurate Biology, China) on a LightCycler 480 (Roche, USA) or ABI QuantStudio 3 system (Thermo, USA). GAPDH and U6 were served as internal standard controls. All assays were replicated three times. The primers used for qRT-PCR were purchased from Accurate Biology (China). The sequences of all primers are listed in Supplementary Table 3.

TRIzol (#15596026, Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from BMMSCs and fresh bone tissues. According to the manufacturer’s instructions, the Biospin miRNA Extraction Kit (#BSC64S1, Bioer Technology, China) was used to extract miRNAs. A ReverTra Ace qPCR RT Kit (#TRT-101, Toyobo, Japan) and Mir-X miRNA First-Strand Synthesis kit (#638315, TaKaRa, USA) were used to synthesize complementary DNA from total RNA and miRNA, respectively. qRT-PCR was conducted in a LightCycler 480 System (Roche, USA) using LightCycler480 software 1.5.1.62 SP3 to determine the mRNA and miRNA expression levels of various genes. GAPDH and U6 were used as internal standard controls for normalization by using the comparative CT method. The sequences of all primers used for qRT-PCR amplification are listed in Supplementary Table S1.