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Dual Luciferase Reporter Gene Technology Service
Dual Luciferase Reporter Assay System for Accurate Gene Expression Analysis
Service Overview
The Dual Luciferase Reporter Assay System is widely used in eukaryotic gene expression and cell physiology studies. Single reporter assays can be influenced by various experimental conditions, and thus, the dual reporter assay is commonly used to enhance the accuracy of experiments. This system utilizes two distinct reporter genes expressed and measured simultaneously within the same system, significantly improving the reliability of experimental results by reducing external influences.
Technical Principle
The system consists of Firefly luciferase and Renilla luciferase, two enzymes capable of catalyzing oxidation reactions that generate bioluminescence. The generated fluorescence signals indicate the level of expression for both enzymes.
Firefly luciferase catalyzes the oxidation of luciferin to produce oxyluciferin, emitting yellow-green bioluminescence at a wavelength of 540-600 nm.
Renilla luciferase catalyzes the oxidation of coelenterazine to produce coelenteramide, emitting blue light at a wavelength of 460-540 nm.
To measure gene expression, the regulatory elements of the target gene are cloned upstream or downstream of the Firefly luciferase gene, creating a luciferase reporter plasmid. After transfection into cells and appropriate stimulation, cell lysis occurs, and luciferase activity is measured. The comparison of the Firefly and Renilla luciferase activities helps assess the impact of the experimental conditions on the target gene's regulatory elements.
Advantages of the Dual Reporter Assay
In Vivo Expression: The assay reflects endogenous gene expression, offering a more accurate representation of in vivo interactions.
High Sensitivity: The dual reporter system minimizes interference from other cellular components.
High Accuracy: The system is effective for both plant and mammalian cells, ensuring consistent results across different biological models.
Applications
miRNA and Target Gene Interaction
This assay is used to explore the regulatory relationship between miRNAs and their target genes. By inserting the target gene sequence (such as 5'UTR, 3'UTR, or ORF regions) into the Firefly luciferase reporter plasmid, miRNA-mediated suppression of luciferase translation is measured.
miRNA and lncRNA/circRNA Interactions
This assay also enables studying the interactions between miRNAs and non-coding RNAs (lncRNAs or circRNAs), which share structural similarities with mRNAs. When miRNA binds to the inserted sequences, it suppresses luciferase translation, leading to reduced fluorescence.
Transcription Factor Activity
The system can measure the activity of transcription factors by inserting promoter sequences into the reporter plasmid. Transcription factors binding to their specific promoter elements will activate luciferase expression, resulting in increased fluorescence.
Promoter Structure and SNP Analysis
Researchers can analyze the activity of promoter sequences by using various promoter truncations or mutations. The system is also useful for analyzing SNPs in promoter regions and their effect on gene activity.
Transcription Factor Activity Verification
Transcription factors can be analyzed by constructing a fusion protein with a GAL4 binding domain. Co-transfection with a GAL4-responsive reporter plasmid allows detection of transcriptional activation or repression.
Sample Requirements
| Application Scenario | Requirements |
| Interaction between miRNA and Target Gene | miRNA sequence information, species information; Target gene sequence information, species information, gene name |
| Targeted Interaction between miRNA and lncRNA/circRNA | miRNA, lncRNA/circRNA sequence information, species information |
| Effect of Transcription Factors on Downstream Genes | DNA sequence information, species information; Target gene sequence information, species information, gene name |
Service Details
Service Process | Working Days | Service Deliverables |
| Customer Order | 10 | 1. Sequencing and validation reports for gene synthesis/vector construction 2. Raw data, result images 3. Standard experimental report (including steps and procedures) |
| Experimental Material Confirmation | ||
| Gene Synthesis/Vector Construction | ||
| Tobacco Injection | 10 | |
| Luciferase Detection | ||
| Quantitative Analysis | 5 | |
| Data Compilation | ||
| Total | 25 |
Case Studies
Tobacco System
For assessing the DNA binding ability of Protein A, we performed a dual luciferase assay in a tobacco transient transformation system. The results showed significantly higher LUC/REN activity for pro-LUC and Protein A compared to the promoter and empty vector combination, confirming that Protein A interacts with the promoter.
Figure 1: Interaction between Protein A and the promoter in tobacco
Dual-luciferase assay in tobacco leaves co-expressing pro-LUC and Protein A, and pro-LUC and an empty vector. Fluorescence activity (LUC/REN) was calculated, and statistical significance was determined using Student's t-test (**P < 0.01).
Frequently Asked Questions (FAQs)
What should I do if the fluorescence value is too high or low?
Fluorescence Too High:
a. Reduce the plasmid transfection amount.
b. After cell lysis, centrifuge and measure the supernatant or dilute the lysate before testing.
Fluorescence Too Low:
a. Optimize transfection conditions to improve transfection efficiency.
b. Ensure cell lysis is effective by controlling the incubation time and the amount of lysis buffer used.
c. Ensure all reagents, including substrates, are stored correctly and used at room temperature for the assay.
How to handle inconsistent replicates? Ensure consistency by using multiple replicates (at least three), optimizing sample preparation, and calibrating pipettes regularly.
Difference Between Firefly and Renilla Luciferase? Firefly luciferase generates yellow-green light (560 nm), whereas Renilla luciferase produces blue light (460 nm), each with specific substrate requirements.
Ready to Explore the Power of Dual Luciferase Reporter Assays?
Unlock the potential of your gene expression analysis with our Dual Luciferase Reporter Assay System. Whether you're studying miRNA interactions, transcription factor activity, or SNP analysis, our reliable and sensitive system can help advance your research.
Contact us today to learn more or to place an order!
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