CircRNA expression vectors can generate trans-spliced products. (A) Schematic representation of circv6GFP.PGK and circv6RLuc.PGK expression vectors. (B) Trans-spliced products expected from circv6RLuc.PGK and circv6GFP.PGK vectors. Only trans-spliced products formed from the CMV (full-length mRNA) and PGK (shorter mRNAs) promoters are shown due to space limitations, but we envisage homotypic (GFP and RLuc) products also being produced, as well as products from trans-splicing of two full-length, CMV-synthesized mRNAs. The relative location of PCR primers (RLuc-F; Rf, RLuc-R; Rr, GFP-F; Gf, and GFP-R; Gr) used for product detection are shown. (C) Endpoint RT-PCR analysis of RNA isolated from 293T cells transfected with the indicated expression plasmids. RNA was isolated 48 h following transfection. (Top panel) PCRs performed on RNA samples that had not been converted to cDNA (−RT). (Bottom panel) RT-PCR products obtained after 25 cycles. (D) RT-qPCR analysis of RNA from 293T cells transfected with the indicated plasmids. The combination of plasmids used in the transfection, as well as primer pairs used in the qPCR, are shown below the graph. The relative abundance of each PCR product was calculated using the ΔΔCt method with GFP or RLuc signal calculated relative to GAPDH. n = 6 ± SD. (E) Sequencing chromatogram of the PCR product obtained from RNA of cells cotransfected with circv6GFP.PGK and circv6RLuc.PGK. PCR primers crossed the GFP(NTD)/RLuc(CTD) exon junction.
