In recent years, reporter gene technology developed based on genetic engineering principles has gradually become one of the most promising tools for gene analysis. Among the many reporter genes developed today, luciferase reporter genes are favored for their rapid detection, high sensitivity, wide linear range, and no endogenous expression in most mammalian cells.
Creative Proteomics has extensive experience in experimental analysis of dual luciferase reporter gene assays to help you design your experiments rationally. We utilize highly efficient transfection reagents and highly sensitive luciferase assay kits to ensure the reliability of the results and provide detailed raw data and experimental reports.
The dual-luciferase reporter gene detection system uses Firefly Luciferase and Rellina Luciferase to form the Dual-Luciferase Reporter Assay. During the experiment, firefly luciferase is related to the specific experimental conditions of gene expression. Renilla luciferase is used as an internal reference for transfection to correct the transfection efficiency of transfection between different samples. Firefly luciferase and Renilla luciferase are biologically active without post-translational modification, and function as reporter genes once translation is complete. By adding a specific luciferase substrate, biofluorescence will be emitted during the reaction between luciferase and the substrate, and the biofluorescence released during the reaction can be measured by a fluorometer.
Compared to traditional co-reporters (e.g., CAT, β-Gal, GUS), the dual-luciferase reporter gene system is more sensitive and is expected to eliminate intrinsic factors that may impair experimental accuracy (such as differences in the number and health of cultured cells), and the detection of reporter genes is extremely fast (complete the test within 30s) and simple.
Gene templates with detailed information on transcription factors, target genes or microRNAs.
The experimental procedure and complete report, including the experimental raw data, pictures, analysis results, etc.
Related vector subcloning
Cell culture and co-transfection of reporter gene plasmids and transcription factors
Luciferase activity assay
Promoter activity verification flow chart
Validation flow chart of the interaction between promoters and transcription factors
Long-term experience in experimental analysis of reporter genes to help you design experiments rationally.
High efficiency transfection reagents and high sensitivity luciferase detection kits to ensure the reliability of results.
Comprehensive technical services, providing detailed raw data and experimental reports.
1. What are the advantages of Luciferase as a reporter gene compared to fluorescent proteins?
Luciferase is more than 10-100 times more sensitive than GFP, and has a wider dynamic range for analysis and comparison. Luciferase does not require fluorescence microscopy, and its fluorescence penetration is higher than that of EGFP and other fluorescent proteins in in vivo experiments, while its background signal is low due to its lack of endogenous activity.
2. The transfection efficiency of the dual-luciferase reporter gene experiment is very low?
If the transfection efficiency is low, it can be improved in three aspects. First, we must ensure that the cell state is good. Usually, we select cells that are in the division phase. Alternatively positive controls can be selected for overexpressed fluorescent protein particles. In addition, the quality of DNA is particularly important, and it is best to verify the enzyme digestion first.
The test results of this experiment are very sensitive, and it is normal to have a certain difference, usually just make sure that it is within an order of magnitude. If the difference exceeds this range, it can be improved in two ways, one is to maintain the uniformity of the sample, and the other is to add samples accurately.
3. Difference Between Firefly Luciferase and Renilla Luciferase
Substrates and cofactors are different:
Firefly luciferase requires luciferin, oxygen, ATP and magnesium ions to emit light; Renilla luciferase requires only coelenterazine and oxygen.
The color of the light is different:
The color of light produced by firefly luciferase is yellow-green with a wavelength of 550-570nm; while Renilla luciferase produces blue light with a wavelength of 480nm. It is precisely because of the different substrate and luminescence colors of these two enzymes that they are widely used in dual-reporter experiments.