A scientific mystery lit up by fireflies -- dual luciferase reporter gene
Do you want to verify the effect of miRNA on target genes? -- Dual luciferase reporter gene assay
Do you want to understand the targeted interaction between miRNA and lncRNA/circRNA? -- Dual luciferase reporter gene assay
Do you want to know what the transcription factor is to a downstream gene? -- Dual luciferase reporter gene assay
Yes, the dual luciferase system is so powerful!
Dual luciferase reporter genes usually use firefly luciferase as the reporter gene and renilla luciferase as the reference gene. The reporter system has the advantages of rapid detection, high sensitivity, wide dynamic range and no intracellular active interference. It is widely used in the research fields of gene regulation and non-coding RNA targeted interaction. Today, we will focus on this topic.
First of all, let's take a look at the background of fluromycin.
Luciferase is a general term for an enzyme in nature that can produce biological fluorescence; By oxidation of the substrate luciferin, fluorescence is produced.
Firefly luciferase is derived from the North American firefly.
Renilla luciferase is derived from the sea kidney.
Although firefly and renilla luciferases have completely different evolutionary origins, their enzyme structures and substrate requirements are completely different. Firefly luciferase acts on beetle luciferin in the presence of ATP, magnesium, and oxygen, while renilla luciferase acts on coelenterin in the presence of oxygen.
What is the principle of dual luciferase to detect the transcriptional regulation of target genes?
The reporter plasmids were co-transfected with the corresponding plasmids, and the cells were lysed after appropriate stimulation or treatment. A firefly fluorescence signal is generated when the firefly luciferase assay reagent is added, representing the firefly luciferase reporter gene (" experimental "reporter gene).
The above reaction was quenched by the addition of renilla luciferase detection reagent, and the renilla luciferase fluorescence reaction (the activity of the "control" reporter gene) was activated simultaneously, and a second measurement was performed simultaneously.
The effects of pre - and post-stimulation or different stimuli on the regulatory elements of interest were judged by the level of luciferase activity.
The advantages of the kit are as follows:
- Rapid detection: from sample lysis to the completion of detection, only 10-15 minutes;
- High sensitivity: Based on the principle of chemiluminescence, luciferase and its substrate bioluminescence system can be very sensitive and efficient to detect gene expression.
- Wide detection range: cell samples with different expression levels can be detected.
- No interference of endogenous activity in cells.
After gold mining in the sand, constantly listening to the voice of users, we collected a number of common problems of dual luciferase reporter genes for you, and laid a solid foundation for subsequent experiments.
Q1: Does this product require the sensitivity of the microplate reader? Do you use a glow or a flash?
A1: This product does not require the sensitivity of the microplate reader, and uses a glow type.
Q2: After adding the firefly fluorescent reagent to detect the value, is the renilla luciferase detection reagent added to the well of the firefly detection reagent? Or do you add the renilla luciferase assay to a new well with cell lysates only?
A2: Detection in the same well, that is, firefly luciferase activity was detected first, followed by renilla luciferase activity.
Q3: If I only want to measure the level of renilla luciferase, can I skip the luciferase step and directly add renilla luciferase working solution after lysis?
Q4: What is the role of renilla luciferase assay substrate?
A4: The action of this substrate not only inhibited firefly luciferase activity, but also detected renilla luciferase activity.
Q5: Does it need to be done on ice after the sample is lysed and added to the steps in the 96-well plate?
A5: If it is not necessary to operate on ice, it is recommended to operate at room temperature.
Q6: What is the difference between Luminometer and fluorescence spectrophotometer?
A6: The sample detected by the fluorescence spectrophotometer cannot emit light by itself; the sample needs to be excited by excitation light of a specific wavelength before it can generate fluorescence and be detected by the fluorescence spectrophotometer. The sample detected by the Luminometer itself can emit light and does not require excitation light for excitation. In other words, the luminometer is an instrument that detects chemiluminescence (fluorescence). Some models of fluorescence spectrophotometers also have the function of luminometer, that is, can also detect chemiluminescence. Whether the fluorometer you are using can be used for chemiluminescence determination please read the instructions of the instrument carefully.
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Abbkine Biotechnology Co., Ltd, founded in 2017, is headquartered in Wuhan, China. The company is committed to research, manufacturing and sales of key reagents in the field of Cell assay and Cell therapy, and has become a key enabler of innovation in the field of cell pharmaceutical globally.