Sybr green pcr master mix kit

Total RNA was extracted from cells using an RNA Purification Kit (EZ Bioscience) in accordance with the manufacturer's protocol, followed by measurement of RNA concentration. For reverse transcription mRNAs of ALP, Runx2, CCAAT/enhancer binding protein α (C/EBPα), CD31 and VEGF, cDNA was synthesized from 1 μg of total RNA using a color reverse transcription kit (EZ Bioscience). Then quantitative PCR was performed using a SYBR Green PCR master mix kit (EZ Bioscience). GAPDH was used as the reference gene, and the primer sequences (BioTNT) of the genes above are listed in Table S1. The following cycling conditions were utilized for RT-PCR: 95℃ for 5 min, followed by 40 cycles at 95℃ for 10 s and 60℃ for 30 s. The relative expression of mRNAs was calculated by the 2^-△△Ct method.

The model establishment process was performed as previously described and is illustrated in Figure 1A. Briefly, specific pathogen-free (SPF) male Sprague-Dawley (SD) rats (weight, 250 g ± 20 g) (n = 15) were intramuscularly injected with methylprednisolone (MPS; Pfizer, Shanghai, China) (20 mg/kg/d) for 3 consecutive days per week for 3 weeks. During the process of model establishment, the morphology of the femoral head, the gene expression of miR-137-3p and Runx2 in the femoral head, and the level of SDF-1α in serum were examined at five time points (Figure 1A). In brief, micro-CT scanning was employed to examine morphological changes of the femoral head during the process. Total RNA was extracted from the femoral head using TRIzol (Invitrogen, Carlsbad, CA, USA). Thereafter, reverse transcription of miR-137-3p was performed by the stem-loop method, while quantitative real-time polymerase chain reaction (qRT-PCR) of miR-137-3p was performed using a SYBR Green PCR Master Mix Kit (EZ Bioscience, Beijing, China). U6 was used as the reference gene of miR-137-3p. The primer sequences (BioTNT, Shanghai, China) are listed in Table S1. The relative expression of miR-137-3p was calculated by the 2^-△△Ct method. The detection methods of Runx2 and SDF-1α were as described in the “qRT-PCR” and “ELISA” sections, respectively.