To analyze gene-miRNA direct interaction, I want to perform a luciferase reporter assay. I plan to clone the 3’UTR of interest in pGL3 basic vector ( which is a promoter-less vector).
Can I use the pRL-TK Renilla Luciferase Control Reporter Vectors (which has a TK promoter), to normalize transfection efficiency?
Thank you
You can use any orthogonal reporter to assess transfection efficiency: Renilla luc (e.g. Rluc8SG), Nan-Ogluc, fluorescent proteins or even fusions of them (FP+luciferase), etc.
The best choice will depend on your particular experimental assay and equipment.
You can also use the next vector in the Promega's line which is pGL4 (pGL4.10 - a promoter-less variety). In pGL4 Promega has tried to remove potential hidden transcription factor binding sites. They have also codon optimized the Ppy firefly luciferase (compared to pGL3: luc(+) --> luc2) which increases the expression level/signal (though, it apparently also increases luciferase mRNA half-life, so inducible reporters have a somewhat delayed time profile).
Thank you Michael Koksharov for your answer and this interesting information. The point is that we already have the pGL3 basic vector and the purchase of other vectors is limited for financial reasons.
In fact, my question is: the use of 2 vectors, one with promoter, and the other without, will have an impact on the results?
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