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Dual-Glo luciferase assay for EV-A71 bicistronic construct

JS Jialei Sun
TY Thinesshwary Yogarajah
RL Regina Ching Hua Lee
PK Parveen Kaur
MI Masafumi Inoue
YT Yong Wah Tan
JC Justin Jang Hann Chu
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EV-A71 bicistronic plasmid with EV-A71 IRES expressing renilla luciferase (RLuc) was used as a measurement for viral protein synthesis, whilst pCMV expressing cap-dependent translation of firefly luciferase (FLuc) served as translation control. This EV-A71 bicistronic plasmid (500 ng) was transfected onto 24-well plate seeded RD cells (1 × 105) using the jet-PRIME transfection system (Polyplus-transfection) according to manufacturer’s instructions. At 12 h post-transfection, cells were treated with various concentrations of gemcitabine (Selleckchem, #S4227) for 12 h. DMSO (0.1%) was used as a negative treatment control and 0.25 mg/mL amantadine was used as a positive treatment control in this assay23. Luciferase activity was detected by Dual-Glo luciferase assay system (Promega, #E2920) using GloMax-Multi detection system. Ratio of FLuc:RLuc was calculated to determine EV-A71 IRES-dependent translation inhibition by gemcitabine.

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