Journal: Nucleic Acids Research
Article Title: 5′ terminal nucleotide determines the immunogenicity of IVT RNAs
doi: 10.1093/nar/gkae1252
Figure Lengend Snippet: IVT-produced 5′-pppA RNAs contain more dsRNA compared with 5′-pppG RNAs. ( A ) Dot blot analysis of IVT short viral and Y5 RNAs was performed with anti-dsRNA J2 antibody, ( B ) then densitometric analysis ( n = 4 for short viral RNA, n = 3 for Y5 RNA) was conducted to assess the level of dsRNA byproducts in IVT-derived RNAs. ( C ) PAGE/Urea analysis was done on short viral RNA and Y5 RNA. ( D , C ) PAGE in native conditions was conducted on IVT control dsRNAs (produced by annealing of sense and antisense RNA strands) and ssRNAs. ( E ) Immuno-northern blot using PAGE native gel followed by transfer to the membrane and detection with anti-dsRNA J2 antibody was used to detect dsRNA. ( F ) PAGE in native conditions of IVT short viral RNAs and semisynthetic short viral RNAs produced by splint ligation revealed the presence of dsRNA in IVT 5′-pppA RNAs but not in semisynthetic counterparts. ( G ) To assess the immunogenic potential of splint-ligated RNAs, an IVT-produced antisense RNA, with or without triphosphate moieties, was added to the sense splint-ligated RNA. The concentration of type I IFN in the supernatants was measured using HEK-Blue IFN assay at 24 h after treatment ( n = 3). The positive control (C(+)) involved transfection with 100 ng/ml 3p-hpRNA, while the negative control (C(−)) was mock-transfected with lipofectamine alone. ( B , G ) Data were compared using two-way ANOVA with Šídák's multiple comparisons test.
Article Snippet: HEK293 cells were transfected with short viral RNA and incubated for 24 h. After incubation total RNA was extracted with Trizol, DNA was removed using DNase I treatment and RNA was purified with GeneJET RNA Cleanup and Concentration Micro Kit (Thermo #K0841).
Techniques: Produced, Dot Blot, Derivative Assay, Control, Northern Blot, Membrane, Ligation, Concentration Assay, Positive Control, Transfection, Negative Control