Mir 23a 3p inhibitor

Synthetic miR-23a-3p mimic (sequence: 5′-ATCACATTGCCAGGGATTTCC-3′), miR-23a-3p inhibitor (sequence: 5′-GGAAATCCCTGGCAATGTGAT-3′) and negative control (NC, sequence: 5′-TTCTCCGAACGTGTCACGTTTC-3′) were purchased from GenePharma (Sunnyvale, CA, USA) along with lentiviral green fluorescent protein (GFP)-tagged vector LV3-pGLV-h1-GFP-puro. The synthetic mRNA (1×10⁹ transducing units/ml) was transfected into BMSCs according to the manufacturer's protocol. The most effective multiplicity of infection (MOI) was decided according to the pilot experiment (data not shown). rBMSCs were plated onto 10 cm dishes at a density of 1×10⁶ cells/dish in 5 ml media and transfected using a GFP-tagged lentiviral vector (lenti-23a-mimic-GFP, lenti-23a-inhibitor-GFP and lenti-GFP) at an MOI of 100 plaque-forming unit/cell in the presence of 5 µl polybrene (Merck KGaA). Fresh media was added at 24 h following transfection. Transfection efficiency was analyzed by calculating the number of GFP tagged cells out of the total number of cells after 72–96 h following transfection. The BMSCs was imaged under a fluorescence microscope (×200). Transfection rate was measured by flow cytometry (9 (link)) (Fig. 2). The BMSCs of the rats were isolated and cultured under standard conditions. The transfection rate was >95%.