Fig. 2. Mouse Oprm1 divergent RT-PCRs amplify sense-stranded circRNAs. (A) Primer maps with locations matching the MOR-1 mRNA and (B) dominant Oprm1 circRNAs are shown. Exons 1–5 are abbreviated e1–e5, and primer labels designate the target exons and orientation (F/R) with respect to transcription. The numbers inside the circles represent circRNA sizes (nt). (C) To confirm that divergent RT-PCR products are generated from circular RNAs, RNAs were pretreated with RNase R, an exonucleolytic enzyme that preferentially digests linear mRNA templates from the 3′ tails but not circular RNAs. Mouse brain total RNAs were digested with (+R) or without (−R) RNase R and samples were split to RT reactions with random hexamer (N6) primers, with (+) or without (−) reverse transcriptase (RTase) enzyme, and then these cDNAs were input to PCR using convergent or divergent primer pairs. (D) Convergent RT-PCR (primers e1F-e3R, lanes 1–4) amplified the linear MOR-1 mRNA, but RNase R predigestion attenuated this amplification. Divergent RT-PCR products (primers e2F-e2R, lanes 5–8) were stable against RNase R pretreatment. Divergent RT-PCR using primers from e1 to e2 (e2F-e1R primers, lanes 9–12) did not generate products. For labels above the gel image: top, middle, and bottom rows of labels represent RNase R treatment, inclusion/omission of RTase enzyme during RT with N6, and the PCR primer pairs used for each sample, respectively. For lane labels below the gel image, L represents the Invitrogen 1 kb plus ladder, and the 1 kb (#) and 500 bp (*) bands are marked. (E) Some circRNAs are known to derive from antisense transcripts overlapped with annotated genes; therefore, to determine whether the observed Oprm1 divergent RT-PCRs derive from sense or antisense circRNAs, RT reactions using strand-specific Oprm1 primers were performed as shown in the scheme. Mouse brain total RNA was incubated with either the Oprm1 e2 sense (e2F) or antisense (e2R) gene-specific primers, with (+) or without (−) RTase enzyme, and amplified with e2 divergent primers. (F) RT with antisense-stranded primer (e2R) can support divergent RT-PCR from sense-stranded circRNAs e2.e3, e2.e3.e5(119) and e2.e3.e4(302) (lanes 3 to 4), but sense-stranded primer (lanes 1 to 2) did not. Labels above the gel, from top to bottom, are for the RT primers, inclusion (+) or omission (−) of RTase enzyme, and PCR primers, while lane labels and size ladders are the same as for (D).