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

Protocol

1. Transfect mRNA to Cells

1. Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent the next day) and incubate overnight in an incubator at 37 °C with 5% CO₂.
2. Infect HeLa cells with vaccinia virus (VACV) at a Multiplicity of Infection (MOI) of 5 or keep uninfected HeLa cells for comparison.
   NOTE: MOI is the number of infectious viral particles per cell.
3. MOI of X= {[(Number of cells * X) / Virus Titer] * 1000} µl of virus per 1 mL medium.
4. After desired h post-infection (hpi) (in this experiment at 10-12 hpi), transfect mRNA (500 ng of total mRNA per well o....

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Results

Figure 1: mRNA synthesis and transfection. (A) Schematic of in vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5'-UTR of interest and the T7 promoter is used as the template. The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5' to 3' direction. Once mRNA is 25-30 nt long, m7G cap is added using an anti-reverse cap analog, ARCA. (B) RNA bands from in vitro

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Materials

Name	Company	Catalog Number	Comments
Corning 96 Well Half-Area white flat bottom polystyrene microplate	Corning	3693	Opaque walled 96 well white plate with solid bottom
Dual-Luciferase Reporter Assay System	Promega	E1960	Dual-Luciferase Assay Kit (DLAK)
GloMax Navigator Microplate Luminometer	Promega	GM2010	Referred as multimode plate reader luminometer
Lipofectamine 2000	Thermo Fisher Scientific	11668019	Cationic lipid transfection reagent
Opti-MEM	Thermo Fisher Scientific	31985070	Reduced serum media