Investigating miRNA-Regulated Gene Interactions

After amplification, circ-FKBP5 and RUNX2 sequences were cloned into pmirGLO plasmids (Promega) to gain circ-FKBp5-wild-type (WT) and RUNX2-WT. A fragment covering the mutant target region was designed and cloned into pmirGLO to gain circ-FKBP5-mutant-type (MUT) and RUNX2-MUT. Subsequently, co-transfection of miR-205-5p mimic or mimic NC with circ-FKBP5-MUT or RUNX2-WT or their respective MUT plasmids was done into BMSCs using Lipofectamine 2000. The relative luciferase activity was analyzed after transfection of 48 h in the luciferase reporter gene assay system (Promega) [26 (link)].

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Shen Y., Jiang B., Luo B., Jiang X., Zhang Y, & Wang Q. (2023). Circular RNA-FK501 binding protein 51 boosts bone marrow mesenchymal stem cell proliferation and osteogenic differentiation via modulating microRNA-205-5p/Runt-associated transcription factor 2 axis. Journal of Orthopaedic Surgery and Research, 18, 782.

Publication 2023

pmirGLO plasmids luciferase reporter gene assay system

Assay Fkbp5 Lipofectamine 2000 Luciferase Plasmids Promega Reporter gene Runx2 Transfection

Corresponding Organization : Changshu Institute of Technology

Other organizations : Nanjing University of Chinese Medicine, Second Affiliated Hospital of Soochow University, Soochow University

Top 5 similar protocols

Variable analysis

independent variables

Co-transfection of miR-205-5p mimic or mimic NC
Circ-FKBP5-MUT or RUNX2-WT or their respective MUT plasmids

dependent variables

Relative luciferase activity

control variables

Transfection using Lipofectamine 2000
Luciferase reporter gene assay system (Promega)

positive controls

Circ-FKBP5-wild-type (WT)
RUNX2-WT

negative controls

Mimic NC

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