A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells
Transcrição
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.
