The target gene of miR-124-3p was predicted using a miRNA bioinformatics software. The TRAF3 3'untranslated region gene fragment was artificially synthesized and introduced into the reporter plasmid pmiR-reporter (Huayueyang Biotechnology Co., Ltd., Beijing, China) with the help of endonuclease sites, Spe I and Hind III. The complementary sequence mutation site of seed sequence was designed based on the TRAF3 WT. T4 DNA ligase was then used to insert the target fragment into the pmiR-reporter plasmid following restriction endonuclease digestion. The luciferase reporter plasmids WT and MUT were co-transfected with miR-124-3p into BNL CL.2 cells. The cells were collected 48 h after transfection, and the protein was extracted. Luciferase activity was tested using a luciferase detection kit (K801-200, BioVision, Palo Alto, USA) with a lomax20/20 luminometer fluorescence detector (Promega Corporation, Madison, WI, USA).
Lomax20 20 luminometer fluorescence detector
Manufactured by Promega
Sourced in United States
The Lomax20/20 is a luminometer fluorescence detector. It is designed to measure luminescence and fluorescence in biological samples.
Automatically generated - may contain errors
3 protocols using lomax20 20 luminometer fluorescence detector
1
Validation of TRAF3 as miR-124-3p Target
2
Transcriptional Activity of KLF5
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The transcriptional activity of KLF5 was determined. The reporter gene plasmids with MKK7 or Slug promoter sequence were co-transfected with KLF5 overexpression or empty plasmid into HEK-293 T cells. After 72 h, the cells were collected and the protein was extracted. A luciferase detection kit (K801-200, BioVision, Mountain View, CA, USA), and a lomax20/20 luminometer fluorescence detector (Promega, Madison, WI, USA) were used to detect luciferase activity. The primer sequences of the MKK7 promoter were as follows: F: 5′-TCGAGCTCTAGGTGGCGTCATCCTT-3; R: 5′-GGGCTGATATCCAGGTTGAGGTCGA-3′; the sequences of the Slug promoter were: F: 5′-TGCGTTCCCAAACCTCACGGA-3′; R: 5′-GCCTTCCCCACAGGCTCCCT-3'.
3
Validating miR-130b Target Gene KLF4
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The StarBase, RNAInter, RNA22, miRanda, and mirDIP databases were used to predict the target genes of miR-130b. The five databases use different binding site matching algorithms, and we thus used the Venn tool to perform an intersection analysis of the prediction results from the five databases. The biological prediction website TargetScan algorithm was then applied to verify whether KLF4 is the direct target gene of miR-130b.
A dual luciferase reporter assay was subsequently used to verify whether KLF4 was the direct target gene of miR-130b. The cVSMCs at the exponential phase were seeded into six-well plates at a density of 3 × 105 cells/well. Upon reaching 70–80% confluence, cells were cotransfected with correctly synthetic luciferase reporter plasmids KLF4 WT and MUT with miR-130b mimic, NC mimic, miR-130b inhibitor, and NC inhibitor using the Lipofectamine 3000 reagent (L3000008, Invitrogen). After 48 h, cells were collected, and the luciferase activity was detected by a luciferase detection kit (K801-200, Biovision) through the Lomax20/20 luminometer fluorescence detector (Promega).49 (link)
A dual luciferase reporter assay was subsequently used to verify whether KLF4 was the direct target gene of miR-130b. The cVSMCs at the exponential phase were seeded into six-well plates at a density of 3 × 105 cells/well. Upon reaching 70–80% confluence, cells were cotransfected with correctly synthetic luciferase reporter plasmids KLF4 WT and MUT with miR-130b mimic, NC mimic, miR-130b inhibitor, and NC inhibitor using the Lipofectamine 3000 reagent (L3000008, Invitrogen). After 48 h, cells were collected, and the luciferase activity was detected by a luciferase detection kit (K801-200, Biovision) through the Lomax20/20 luminometer fluorescence detector (Promega).49 (link)
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