Dual-Luciferase Assay for Influenza Protein Expression

Subconfluent monolayers of 293T cells (7.5 × 10⁵ cells in 35-mm dishes) were transfected with the luciferase reporter plasmid (enhanced green fluorescent protein [EGFP] open reading frame in pHW72-EGFP replaced with the firefly luciferase gene) [45 (link)] and a mix of PB2, PB1, PA and nucleoprotein (NP) expression plasmids (CA/04 or mutated) in quantities of 1, 1, 1, and 2 μg, respectively. The plasmid pGL4.75(hRluc/CMV), which expresses Renilla luciferase (Promega, Madison, WI, USA), was used as an internal control for a dual-luciferase assay. As a negative control, 293T cells were transfected with the same plasmids, with the exception of the NP expression plasmid. After 24 h of incubation at 37°C cell extracts were harvested and lysed, and luciferase levels were assayed with a dual-luciferase assay system (Promega) and a Synergy 2 multimode microplate reader (BioTek Instruments, Winooski, VT, USA). Experiments were performed in triplicate.