Introduction
Firefly luciferase and Renilla luciferase are called reporter genes because the ratio of the fluorescence generated by their expressions can be used to monitor and confirm the intermolecular interactions at the microscopic level in the study of gene expression regulation. The specific approach is as follows: Clone the transcriptional regulatory elements or the 5’ promoter region of the target gene upstream of the Firefly luciferase gene, or clone the 3’-UTR region or the lncRNA binding sequence downstream of the Firefly luciferase gene to study the strength of the promoter, the effect of transcription factors on the promoter, or the regulatory effect of miRNA on the target gene or lncRNA (see Figure 2). Meanwhile, introduce the TK vector containing the Renilla luciferase gene as an internal reference in order to eliminate the inter-group errors caused by factors such as cell transfection.
1) Promoter Region Binding Study
Clone the transcriptional regulatory elements or the 5’ promoter region of the target gene upstream of the Firefly luciferase gene, and then co-transfect them with the transcription factor expression plasmid into the tool cells. Detect the expressions of Firefly luciferase and Renilla luciferase in the cells. The expression of Renilla luciferase is used as an internal reference to eliminate the inter-group errors caused by factors such as cell transfection. If the relative fluorescence value of Firefly luciferase/Renilla luciferase in the group transfected with the 5’ promoter region sequence is significantly increased, while there is no change in the group with the mutated 5’ promoter region sequence, it indicates that the 5’ promoter region sequence is the promoter region of the transcription factor and mainly binds through the mutation sites.
2) 3’UTR Region Binding Study
Clone the 3’-UTR region or the lncRNA binding sequence downstream of the Firefly luciferase gene, and then co-transfect them with miRNA into the tool cells. Detect the expressions of Firefly luciferase and Renilla luciferase in the cells. The expression of Renilla luciferase is used as an internal reference to eliminate the inter-group errors caused by factors such as cell transfection. If the relative fluorescence value of Firefly luciferase/Renilla luciferase in the group transfected with the 3’-UTR region or the lncRNA binding sequence is significantly decreased, while there is no change in the group with the mutated 3’-UTR region or the lncRNA binding sequence, it indicates that the 3’-UTR region or the lncRNA binding sequence is the miRNA binding region and mainly binds through the mutation sites.
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