Service Introduction:
The luciferase reporter gene system uses luciferin as a substrate to detect luciferase activity. Luciferase can catalyze the oxidation of luciferin to oxyluciferin. In the process of luciferin oxidation, it will emit bioluminescence. The bioluminescence released during the oxidation of fluorescein can then be measured by a fluorescence analyzer (chemiluminescence). Luciferin and luciferase, a bioluminescence system, can detect gene expression extremely sensitively and efficiently.
Dual luciferase system, that is, there are two kinds of luciferase, the more common combination is firefly luciferase and Renilla luciferase, firefly luciferase acts on beetle luciferase, and Renilla luciferase exists in the presence of oxygen Acting on Renilla luciferin. Using one of them as an internal control, that is, the expression level is basically constant, is used to reduce the inherent changes between different treatments in the experiment, including the difference in the number and viability of cultured cells, the efficiency of cell transfection and lysis, and the other luciferase It is used as a reporter gene to indicate gene expression under different processing conditions.
Application method:
1. Identify the homeopathic original or trans-acting factor;
2. Identify the target gene of miRNA: miRNA binds to the 3'UTR region of target mRNA through base pairing, inhibits translation of target gene or target gene mRNA The degradation can achieve the purpose of regulating gene expression. Construct the 3'UTR region of the target mRNA to the 3'end of the firefly luciferase reporter gene, co-transfect the cells with the miRNA expression vector and the Renilla luciferase reporter gene vector, and add two substrates of luciferin successively. The detection of luciferase reporter gene expression confirms the regulatory effect of miRNA on the target gene.
