Our protocol for an in vitro transcribed RNA-based luciferase assay helps study translation regulation in pox virus infected cells. Additionally, a customized length of the Poly(A)liter allows studying its regulatory effects on translation. The main advantage of this technique is that it allows for the study of translation regulation by Cis elements such as 5'UTR and 3'UTR in mRNA and broad spectrum of translation initiation.
The in vitro transcribed RNA-based luciferase reporter assay may be tailored to incorporate modification to cap and other modification and used to test the effect on translation. To begin, design forward and reverse primers to generate PCR amplicon containing the following elements in 5'to 3'direction:T7-Promoter, Poly(A)liter, Firefly Luciferase ORF in a Poly(A)tail referred to as T7_12A-Fluc, include several extra nucleotides in forward primer followed by T7-Promoter Poly(A)liter or desired 5'UTR sequence and approximately 20 nucleotides corresponding to the 5'end of the reporter gene's ORF. Adjust the primer length based on melting temperature corresponding to the 5'end of the reporter gene's ORF ensuring that the corresponding region in the primer is identical to the sense strand of the gene.
Then design reverse primer to include Poly(A)tail and approximately 20 nucleotides. Adjust based on melting temperature corresponding to the 3'end of the reporter gene's ORF ensuring that the corresponding region of the primer is identical to the antisense strand of the gene and an in-frame stop codon is present before the Poly(A)tail. For internal control, design another set of primers containing the following elements in 5'to 3'direction:T7-Promoter, a random 5'UTR coding sequence containing Kozak sequence, Renilla Luciferase ORF, and Poly(A)tail, referred to as T7_Kozak-Rluc.
To set up the reaction, add reagents in the following order to a PCR tube:DNase free water, 2X high-fidelity DNA polymerase, primers and sequence confirmed luciferase template DNA. After a standard three-step PCR cycle to generate a DNA template, detect the PCR product by running 5-10%of the PCR reaction in a 1%agarose TAE gel electrophoresis along with a molecular weight standard. To determine the size of the PCR product, visualize the gel under a UV illuminator.
After determining the correct size of the PCR product, purify it using a commercially available PCR purification kit using 100 microliters of nuclease free water to elute the DNA. Check the concentration of the purified DNA using a spectrophotometer and store it at minus 20 degrees Celsius for use for in vitro transcription immediately. To synthesize RNA from the PCR product, add the following reagents to a microcentrifuge tube:DNase-RNase free water, NTP buffer mix, cap analog, PCR product template, and T7-RNA polymerase mix from in vitro transcription kit.
Mix thoroughly. Incubate at 37 degrees Celsius for two hours and then proceed to the purification of the synthesized RNA using an RNA purification kit. To check the purified RNA, heat RNA at 70 degrees for five minutes to remove secondary structure and run it on 1.5 agarose TBE gel.
Then visualize the gel under a UV illuminator. Check the concentration of the purified RNA using a spectrophotometer. Aliquot it and store at minus 80 degrees Celsius.
Seed HeLa cells in a 24 well plate to achieve 80-90%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 five 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 five 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 drop wise and evenly per well of the 24 well plate. Five 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 four 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 and a multi-mode plate reader luminometer to measure the dual luminescence using kinetics function as described in the manuscript.
After exporting the luminescence reading data into a desirable file format, determine relative translation rate from 12A Fluc mRNA in uninfected and VACV infected HeLA cells by dividing Fluc value by internal control Rluc value. This assay was used to test the translation efficiency of an Fluc mRNA that contains a 5'Poly(A)liter in uninfected and VACV infected cells. Both uninfected and VACV infected cells were successfully co-transfected with Fluc and Rluc mRNA.
Dividing Fluc by Rluc normalized the transfection efficiency in RNA stability in cells. 5'Poly(A)liter containing mRNA has a translational advantage during VACV infection compared to uninfected cells. This was not due to differential transfection efficiency or mRNA stability as the RNA level was similar in uninfected and VACV infected cells five hours post mRNA transfection.
Take special care when designing primers as this can affect not only PCR reaction efficiency but also all downstream processes requiring the amplified DNA. This method is an important assay to quickly test the translation regulation by Cis elements. If possible, this method should be corroborated by other complementary experiments.
Precaution should be taken while using Vaccinia virus and avoid exposure to chemicals such as ethidium bromide and phenol.
