Dual luciferase reporter assay
Dual luciferase reporter assay
Summary
The target gene transcriptional regulator original is constructed onto the firefly luciferase reporter gene plasmid, and co-transfected with the sea kidney luciferase endogenous reference gene plasmid to transfect cells, which will emit biofluorescence after lysis of the cells and addition of the luciferase substrate, respectively, which will be read by the fluorometer.
Calculation of the relative ratio of firefly luciferase/sea kidney luciferase can determine the effect on the transcriptional regulatory progenitor of the target gene.
Operation method
Dual luciferase reporter gene assay
Principle
The target gene transcriptional regulator original is constructed onto the firefly luciferase reporter gene plasmid, and co-transfected with the sea kidney luciferase endogenous reference gene plasmid to transfect cells, which will emit biofluorescence after lysis of the cells and addition of the luciferase substrate, respectively, which will be read by the fluorometer. Calculation of the relative ratio of firefly luciferase/sea kidney luciferase can determine the effect on the transcriptional regulatory progenitor of the target gene.
Materials and Instruments
Luciferase reporter gene and internal reference gene plasmid, PBS, cell lysate, firefly luciferase substrate
Termination solution (containing sea kidney luciferase substrate) (Promega E1910)
Move
(1) Plasmid construction and transfection:
The firefly luciferase reporter gene with the target sequence was constructed, and the cells were co-transfected with the plasmid of the sea kidney luciferase internal reference gene, and the samples could be harvested 24 h later.
(2) Lysis:
Cells were washed twice with PBS, then appropriate amount of cell lysate was added (original solution was 5X, diluted to 1X with double-distilled water; reference volume: 100~150 μL for each well of 24-well plate, or according to the following table, appropriate amount of lysate was added), cells were blown up repeatedly, and lysed for 15 min by shaking, then the cells were collected into 1.5 mL EP tubes, and the cells were centrifuged at 10,000 g at 4 ℃ for 10 min, and the supernatant was taken into new EP tubes. Collect the cells into a 1.5 mL EP tube, centrifuge at 10000 g for 10 min at 4 ℃ and remove the supernatant into a new EP tube.
(3) Configure the reaction substrate:
① Configure substrate 1 (S1) for firefly luciferase: Configure the substrate when the kit is used for the first time, add the substrate buffer into the brown bottle containing solid substrate, dissolve and shake well, and store at -80 ℃ under light protection according to the quantity required for each experiment (e.g., 1 mL/EP tube), and then dissolve it at room temperature, also under light protection, when it is used. Calculate the number of samples and dispense one sample per 50 μL into EP tubes.
② Configure stop&Glo (S2) substrate for kidney luciferase: add 1 μL of stop substrate to every 50 μL of stop buffer, and configure as required according to the number of samples, one sample for every 50 μL of assay.
(4) Measurement reading:
Take 10 μL of the sample to be tested and add it to 50 μL of the reaction substrate S1, gently blow several times with a pipette (be careful to avoid air bubbles), and then quickly put it into the instrument for measurement (EP tube with open cap), and then measure the first value (firefly luciferase activity S1).
After completion, the EP tube was quickly removed, 50 μL of stop&Glo liquid was added, gently blown with a pipette to mix, and quickly placed in the instrument to obtain the second value (sea kidney luciferase activity S2).
After the above operation, the ratio of the two values displayed by the instrument is the required data.
Caveat
(1) Firefly luciferase substrate should be divided into portions to avoid repeated freezing and thawing, and stored at -80 ℃ to avoid light, and kidney luciferase substrate should be prepared and used now.(2) Before the experiment, each component (cell lysis product, substrate working solution, etc.) needs to be returned to room temperature, and pay attention to the experimental process to avoid light, the whole process should be completed within 30 min, each sample should be fully mixed, and the number of times and time of mixing should be consistent as far as possible.(3) If the cell lysis products need to be stored temporarily, not more than 6 hours at room temperature, -20 ℃ for one month, -80 ℃ for six months.
Common Problems
(1) The measured fluorescence value is low. First, to rule out transfection efficiency problems, spread one more well of 12-well plate with cells transfected with fluorescent plasmid, observe the fluorescence efficiency of transfection, and set up positive and negative controls. After eliminating the transfection problem, if the fluorescence reading is still low, try to increase the amount of transfected plasmid.
(2)The measured fluorescence value is too high. Reduce the amount of transfected plasmid, or appropriately dilute the supernatant after cell lysis and centrifugation.
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