This article describes a method for measuring luciferase activity in mammalian cells using firefly and Renilla luciferase reporter mRNAs. The protocol involves transfecting cells with these mRNAs and analyzing luminescence to assess the effects of poxvirus infection on luciferase production.
Take transfection complexes comprising reporter mRNAs encoding for firefly luciferase, or Fluc, and Renilla luciferase, or Rluc.
Fluc mRNA contains a 5' polyadenosine sequence, or a poly(A) leader, while Rluc mRNA lacks this sequence.
Transfect uninfected and poxvirus-infected mammalian cells to deliver the reporter mRNAs, and incubate.
In uninfected cells, eukaryotic initiation factors bind to the mRNA's 5' cap, followed by recruitment of the small ribosomal subunit and an initiator tRNA.
Upon the small subunit locating the start codon, the large subunit joins to synthesize luciferase proteins.
In infected cells, poxvirus-mediated phosphorylation of the small subunit causes ribosome assembly directly on the leader sequence, enhancing Fluc production.
Lyse the cells to release the luciferases.
Add the Fluc substrate, which the enzyme oxidizes, emitting light. Using a plate reader, measure the luminescence.
Repeat the step for Rluc to determine the relative increase in the Fluc luminescence compared to Rluc in virus-infected cells.
Seed HeLa cells in a 24-well plate to achieve 80% to 90% confluency the following day, and incubate overnight in an incubator at 37 degrees Celsius with 5% carbon dioxide. Infect HeLa cells with Vaccinia virus at a multiplicity of infection of 5 and keep uninfected HeLa cells for comparison, then incubate the plate at 37 degrees Celsius with 5% carbon dioxide for 10 to 12 hours. After desired hours post-infection, mix 480 nanograms of 12A sequence-bearing firefly luciferase mRNA and 20 nanograms of Kozak sequence-bearing Renilla luciferase mRNA in one microcentrifuge tube.
In another microcentrifuge tube, add 1.1 microliters of cationic lipid transfection reagent. Add 55 microliters of reduced serum media to both tubes. Mix and incubate at room temperature for 5 minutes. Then, add 55 microliters cationic lipid transfection reagent containing reduced serum media to mRNA-containing tube. Mix gently but thoroughly with a pipette and incubate at room temperature for 15 minutes. During the incubation, remove the cell culture medium from the cells and add 400 microliters of reduced serum medium per well.
When incubation is completed, add 100 microliters of the mixture dropwise and evenly per well of the 24-well plate. 5 hours post-co-transfection with 12A-Fluc and Kozak-Rluc mRNA, remove the reduced serum medium from the cells, and lyse the cells by adding 150 microliters 1X lysis buffer from the luciferase assay kit capable of performing two reporter assays. After incubating for 10 minutes at room temperature, scrape the cells using rubber and sterile syringe and transfer to a microcentrifuge tube.
To pellet cell debris, centrifuge the lysate at 12,000 times g for 10 minutes at 4 degrees Celsius. Transfer 30 microliters of supernatant per well of an opaque-walled 96-well white assay plate with a solid bottom. Use the luciferase assay kit in a multi-mode plate reader luminometer to measure the dual luminescence using kinetics function as described in the manuscript.
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