3′-UTR of the GRM5 or LINC00314 gene containing putative miR-129-5p targeting site was amplified by chemical synthesis and was inserted into the psiCHECK2 vector (Promega, Madison, WI, USA). When the confluence was up to 70%, ADSCs were transfected with related mixtures including 50 ng GRM5/LINC00314 wild-type or GRM5/LINC00314 mutant-type 3′-UTR reporter plasmids, miR-129-5p mimics, or miR-129-5p NC in a final concentration of 20 nM, and Lipofectamine 2000 for 48 h. Luciferase activity was detected using the dual-luciferase reporter gene kit (Beyotime, Shanghai, China).
Dual luciferase reporter gene kit
Manufactured by Beyotime
Sourced in China
The Dual-luciferase reporter gene kit is a tool used for the simultaneous measurement of the activities of two different luciferase reporter enzymes within a single sample. It provides a reliable and sensitive method for analyzing gene expression and regulatory mechanisms.
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5 protocols using dual luciferase reporter gene kit
1
Validating miR-129-5p Targeting of GRM5 and LINC00314
2
Keap-1 3'-UTR Luciferase Assay
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The HCMs were first inoculated into 12-well plates at a density of 1×105 cells per well. The 3'-untranslated region (3'-UTR) of the Keap-1 gene containing putative miR-129-5p targeting site was amplified by chemical synthesis and was inserted into the psiCHECK2 vector (Promega, Madison, WI, USA).When the confluence reached 70%, the HCMs were transfected with related mixtures, including 50 ng keap-1 wild-type or keap-1 mutant-type 3'-UTR reporter plasmids, miR-129-5p mimics or miR-129-5p NC in a final concentration of 20 nM, and Lipofectamine 2000 for 48 hours. Luciferase activity was detected using the dual-luciferase reporter gene kit (Beyotime, Shanghai, China).
3
Validating miR-124-3p Targets XIST and IRF1
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The starbase database predicted that there were targeted binding sites between miR-124–3p and 3′ untranslated region (UTR) of XIST, IRF1. The targeting relationship was further verified by dual-luciferase reporter experiment. The wild-type (WT)/mutant (MUT) plasmid of XIST and the WT/MUT plasmid of IRF1 were transfected into BV2 cells together with NC mimic and miR-124–3p mimic, respectively. After culture for 48 h in a cell incubator, the luciferase activities were measured according to the instructions of the dual-luciferase reporter gene kit (Beyotime, Shanghai China). Renal luciferase activity was served as internal reference.
4
Regulatory mechanism of RUNX2 by miR-505
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MC3T3-E1 cells were firstly inoculated into 12-well plates at a density of 1 × 105 cells per well. 3′ UTR of the RUNX2 gene containing putative miR-505 targeting site was amplified by chemical synthesis and was inserted into the psiCHECK2 vector (Promega, Madison, WI, USA). When the confluence was up to 70%, MC3T3-E1 were transfected with related mixtures including 50 ng RUNX2 wild-type or RUNX2 mutant-type 3′ UTR reporter plasmids, miR-505 mimics or miR-505 mimics NC in a final concentration of 20 nM, and Lipofectamine 2000 for 48 h. Luciferase activity was detected using the dual-luciferase reporter gene kit (Beyotime, Shanghai, China).
5
Luciferase Assay for CX3CL1 Promoter
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GC cells were seeded onto a 96-well plate at the concentration of 3,000 cells per well for 24 h of culture. The cells were transfected with CX3CL1 promoter sequence plasmids (Genechem, Shanghai, China) persistently expressing firefly luciferase. The pRL-TK plasmids persistently expressing Renilla luciferase (the control) were used to evaluate the transfection efficiency. A dual luciferase reporter gene kit (Beyotime, China) and a multifunctional enzyme labeling instrument (Molecular Devices, USA) were used to detect the luciferase activity of each group.
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