Principle and application of dual luciferase reporter gene detection kit (dual luciferase activity)
Event preview:At the end of the article, there is a lottery activity recommended by Li Ji's Biologics Firefly&Renilla dual luciferase reporter gene testing kit. The rewards include Tencent members, Music members, etc. The lottery will be held on June 28th. Come and participate!
Recommended dual luciferase products from Heyuan Liji
- Experimental principle -
Firefly luciferase is a protein with a molecular weight of approximately 61KD, which can catalyze the oxidation of Luciferin to Oxylluciferin in the presence of ATP, magnesium ions, and oxygen. During the oxidation process of Luciferin, it emits biofluorescence.
Figure 1. Principle of firefly luciferase catalyzed reaction
Sea kidney luciferase is a protein with a molecular weight of approximately 36KD, which can catalyze the oxidation of Coelenterazine to Coelenteramide in the presence of oxygen. During the oxidation process of Coelenterazine, it also emits biofluorescence.
Figure 2. Principle of Sea Kidney Luciferase Catalytic Reaction
The dual luciferase reporter gene detection system simultaneously expresses firefly luciferase and sea kidney luciferase in cells, and firefly luciferase acts as aReporter geneChanges in the expression of target genes in response to sea kidney luciferaseInternal reference gene，Provide a comparison of transcriptional efficiency (reducing the impact of intrinsic factors such as the number of cultured cells, the efficiency of cell transfection and lysis on experimental accuracy). The two luciferases do not have species homology and correspond to different reaction substrates, therefore there is no cross interference.
- Application direction -
1、 Target gene validation
MiRNA mainly acts by degrading or inhibiting translation by acting on the 3'UTR of the target gene. The 3'UTR (wild-type and binding site mutant) sequence of the target gene is constructed into the 3 'end of the reporter gene Luc in the vector. By comparing the changes in gene expression after overexpression of miRNA (whether the activity of luciferase decreases or remains unchanged), the role of miRNA and the interaction site between miRNA and the target gene 3'UTR are clarified.
Figure 3. Design principle of target gene validation experiment
Applicable types of related experiments: miRNA target genes (mRNA, LncRNA, circRNA), m6A sites, enhancers
2、 Validation of promoter activity
Transcription factors mainly act by acting on the promoter of the target gene. The promoter region sequence of the target gene is constructed at the 5 'end of the reporter gene Luc. After co expression of transcription factors, the binding site of transcription factors with the target gene promoter and their effect on the target gene are determined based on changes in the expression of the reporter gene.
Figure 4. Design principle of promoter validation experiment
- Application examples -
1. Example 1
Article Title：METTL14/miR-29c-3p axis drives aerobic glycolysis to promote triple-negative breast cancer progression though TRIM9-mediated PKM2 ubiquitination
Publishing journals：Journal of cellular and molecular medicine
Heyuan Assistance: WT TRIM9-3'UTR and mutant TRIM9-3'UTR were constructed into the pMIR-REPORT vector, and the binding between miR-29c-3p and TRIM9 was clarified by co transfection with miR-29c-3p mimics, miR-29c-3p inhibitor, and miRNA-NC.
2、Example 2
Article Title：The ELAVL3/MYCN positive feedback loop provides a therapeutic target for neuroendocrine prostate cancer
Publishing journals：Nature communications
To elucidate the molecular mechanism of MYCN transcriptional regulation, the authors constructed the full length of ELAVL3 and the promoters of Segments A, B, and C onto the luciferase reporter gene vector. Through dual luciferase reporter gene experiments, it was found that MYCN significantly upregulated the luciferase signals of the full length ELAVL3 and ELAVL3 Segment A groups, proving the transcriptional regulation of ELAVL3 by MYCN.
3、Example Three
Article Title：METTL3 facilitates stemness properties and tumorigenicity of cancer stem cells in hepatocellular carcinoma through the SOCS3/JAK2/STAT3 signaling pathway
Publishing journals：Cancer gene therapy
Heyuan Assistance: The CDS region of SOCS3, which includes the m6A recognition site wild-type (GGACU) and mutant (CCTGA), was constructed on the pMIR REPORT vector. The interaction between METTL3 and SOCS3 was confirmed through overexpression and interference with METTL3 expression, and dual luciferase reporter gene experiments.
/Dual luciferase activity/
Event time: From today to June 28th
Prizes（randomExtract 6 digits）：Send a one-year annual card, including iQiyi, Tencent Video, Youku, Mango, Bilibili, QQ Music, and NetEase Cloud Music-(SelectableOne of them)
Activity scope: National biological relatedEnd customers
Lottery opening time: June 28, 2024 at 13:00
Claim method:3 working daysInternal contactManager LiangRedemption, overdue will be considered as automatic abandonment
matters needing attention:
Reply after following WeChat official account“Dual luciferase”Participate in activities；
Draw to send iQiyi, Tencent Video, Youku, Mango, Bilibili, QQ Music, NetEase Cloud Music-One of them；
Can be transferred, overdue will be considered as automatic abandonment;
Little cute people can follow the official account to get the latest activities;
The final interpretation of the activity belongs to Heyuan Li Ji.
Back
