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. 2010 Jan;16(1):170–185. doi: 10.1261/rna.1873910

FIGURE 4.

FIGURE 4.

Detection and confirmation of biological activity of the γHV68 miRNAs during lytic infection in vitro. (A) RLM-RT-PCR for miRNAs in 3T3 cells infected with either γHV68 or γHV68Δ9473. mmu-miR-21 serves as an endogenous control for miRNA expression in 3T3 cells. RT-PCR products were resolved by gel electrophoresis and visualized by ethidium bromide. (B) RT-PCR for ORF 50, a marker of lytic infection for 3T3 cells infected with γHV68. Actin serves as a RT-PCR control for endogenous transcription in 3T3 cells. (C) Dual luciferase reporter analysis following infection with either the γHV68 or γHV68Δ9473 viruses. pGL3 luciferase reporter target is labeled below the graph. All results are displayed as a ratio of firefly luciferase expression in γHV68 infected cells divided by firefly luciferase expression in γHV68Δ9473 infected cells. Firefly luciferase readings are all normalized to the renilla luciferase transfection control. Readings obtained from 3T3 infected cells are shown in solid black bars; 293 cells, in solid white bars. Readings below the dashed line represent repression by the corresponding viral miRNA in γHV68 infected cells.