| Product Description | Product introduction： Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity. Instruction：
1.Cell lysis
( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. 
Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ).
( 2 ) The pyrolysis products were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection.
Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection.
2. Preparation of working fluid
( 1 ) Restore all components to room temperature.
( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution.
Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use.
( 3 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 μL E component was added to the 49 μL D component.
Note : Renilla luciferase working solution needs to be prepared now.
3.chemiluminescence value detection
( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s.
( 2 ) each sample determination, take the sample 20-100 μL ( if the sample volume is enough, please add 100 μL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control.
( 3 ) 100 μL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ).
Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution.
( 4 ) 100 μL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ).
( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.
Component： 
Recommendation： It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.
Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves. Scope of application： Study on gene expression regulation and promoter |
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