Reporter assays
Firefly luciferase reports the element of interest and Renilla luciferase, from a co-transfected plasmid with a weak constitutive promoter, normalises it. Both are read in the same lysate: firefly first, then a reagent that quenches it and starts the Renilla reaction.
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Dual luciferase
Firefly luciferase reports the element of interest and Renilla luciferase, from a co-transfected plasmid with a weak constitutive promoter, normalises it. Both are read in the same lysate: firefly first, then a reagent that quenches it and starts the Renilla reaction.
How it differs from the standard method
- Use a weak promoter (TK) for Renilla, at 1:10 to 1:50 of the firefly plasmid.
- Check raw Renilla across conditions: it must not move with the treatment.
- Read with injectors so every well is read at the same time after reagent addition.
- Include a minimal-promoter or empty firefly vector.
1Borrowing a promoter's voice
A reporter construct puts a DNA element you care about (a promoter, an enhancer, a response element such as five tandem NF-κB sites) in front of a gene whose product is easy to measure. When transcription factors act on the element, the reporter is transcribed, translated and accumulates, and the measurement stands in for the element's activity.
Light from a reaction
Firefly luciferase oxidises luciferin with ATP; Renilla luciferase oxidises coelenterazine without ATP. Lysates are read one after the other in the same well, with near-zero background.
Light from the protein
No substrate: read live cells, repeatedly, by plate reader, microscope or flow. Needs time to fold and mature, and competes with cellular autofluorescence.
Colour from an enzyme
β-galactosidase cleaves ONPG to yellow o-nitrophenol (or X-gal to blue in fixed cells). Cheap and robust, but long-lived and shadowed by the cells' own lysosomal enzyme.
2Normalisation and controls
In a transient transfection each well takes up a different amount of plasmid, so a second, constitutively expressed reporter is co-transfected and the experimental signal is divided by it: firefly over Renilla in the dual-luciferase format. That only works if the control reporter ignores the treatment.
3The reporter's half-life sets what you can see
What you measure is reporter protein, which integrates past promoter activity over roughly its own lifetime. Firefly luciferase (half-life of a few hours) and destabilised GFPs carrying a PEST degron (about two hours) follow a transient pulse.
Key terms
Practise these as flashcards →A short DNA sequence bound by a specific transcription factor, often repeated in tandem in front of a minimal promoter.
A co-transfected, constitutively expressed second reporter used to correct for transfection efficiency and cell number.
Normalised signal of the treated condition divided by that of the untreated one.
Relative light units: luminometer counts, comparable only within one instrument and setting.
A reporter fused to a degradation signal (e.g. PEST) so its half-life is short and it follows current promoter activity.
A TATA box and start site with no enhancers; on its own it gives low basal transcription.
When to use it
- To ask whether a pathway or transcription factor is activated by a stimulus, with a quick, quantitative and scalable readout.
- To map which part of a promoter or enhancer is needed for a response, by comparing mutated constructs.
- To screen compounds or genetic perturbations for pathway activation or inhibition in a stable reporter line.
Compared with alternatives
Measures the endogenous gene's mRNA directly, in its native chromatin, at the cost of throughput.
Shows the pathway's own proteins (phosphorylation, degradation of an inhibitor) rather than a downstream proxy.
The instrument side: gain, integration time, plate choice and linear range for luminescence and fluorescence.
How the reporter DNA gets into the cells; efficiency and toxicity drive most of the well-to-well variation.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Firefly luciferase reports the element of interest and Renilla luciferase, from a co-transfected plasmid with a weak constitutive promoter, normalises it. Both are read in the same lysate: firefly first, then a reagent that quenches it and starts the Renilla reaction.
- Use a weak promoter (TK) for Renilla, at 1:10 to 1:50 of the firefly plasmid.
- Check raw Renilla across conditions: it must not move with the treatment.
- Read with injectors so every well is read at the same time after reagent addition.
- Include a minimal-promoter or empty firefly vector.