Figure 1. Isolation of immunostimulatory RV RNAs from in vitro-transcriptions.

(A) In vitro-transcription (IVT) reactions were carried out using purified RRV DLPs, α³²P-GTP, S-adenosylmethionine, and all four NTPs (complete), all reaction components except ATP (incomplete), or all reaction components except DLPs (no DLPs). Reactions were carried out as described in the methods and analyzed on a 20% urea-acrylamide gel. (B) Complete and incomplete IVT reactions were carried out and total RNA was purified from the reactions and analyzed on a denaturing urea-acrylamide gel. UV shadowing was used to visualize large and small molecular weight (MW) RNAs, which were then excised from the gel and purified. Huh7 human hepatoma cells were co-transfected with pIFN-beta-luc (firefly luciferase) and pRL-TK (Renilla luciferase). After 24 hours (hr), cells were mock-transfected, transfected with the entire recovered large or small MW RNA sample, or transfected with 500 ng/well of polyI:C in duplicate. Approximately 21 hr later, cell lysates were made and analyzed using the dual-luciferase reporter assay system. The firefly luciferase light unit values were divided by the Renilla light units (transfection efficiency control) to generate the relative luciferase (luc) activity value. Bars show the average relative luc values plus standard deviation.