Ariamx real time quantitative pcr machine

Total RNA was extracted from OSCC tissue and ANT using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The cDNA was generated in the T100 gradient PCR machine (Bio-rad, USA) using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). RT-qPCR was performed using TB Green Premix Ex Taq II Kit (Takara, Japan) on an AriaMx Real-time quantitative PCR machine (Agilent, USA). The PCR reaction conditions were 95℃ for 30 s, followed by 40 cycles at 95℃ for 5 s and 60℃ for 30 s. Each reaction was performed in triplicate. The expressions of the angiogenic genes including ANG, bFGF, EGF, HGF, HB-EGF, VEGF, PDGF-BB, Leptin, and MMPs including MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, TIMP-2, and TIMP-4 were analyzed by RT-qPCR. GAPDH gene was used as the internal control. The fold change relative to the control group was measured by the 2^−∆∆Ct method. Primer sequences used in this study are listed in Table S2.

The femurs were collected from wild-type, Mir155-Tg, and Mir155-KO mice. And bones were ground with a high-speed low-temperature tissue homogenizer (Servicebio, China). As previously reported (Teng et al., 2022 (link)), the miRNAs were extracted from BMSCs and ground bone tissues with the MolPure Cell/Tissue miRNA Kit (Yeasen, China) as per the manufacturer’s instructions. The miRNA was further reversed by Tailing reaction using miRNA first strand cDNA synthesis kit (Accurate Biology, China). In brief, 3.75 μL miRNA, 5 μL 2×miRNA RT Reaction Solution, 1.25 μL miRNA RT Enzyme Mix were incubated at 37°C for 1 hr, 85°C for 5 min. The mRNAs from osteoclasts induced from Mir155-KO and wild-type mice were extracted with an RNA extraction kit (Accurate Biology, China) as per the manufacturer’s instruction. Total RNA (500 ng) was transcribed with reversed regents (Accurate Biology, China). RT-qPCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, China) on an AriaMx Real-time quantitative PCR machine (Agilent, USA). The PCR conditions were 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. The fold change relative to the control group was measured by the 2^-∆∆Ct method. The primers used were shown in Table 1.