Figure 3.

Biological experiments. (a) Schematic representation of divergent primer design and its interests. This primer pair enables amplification not only of circRNA but also of linear RNA when the latter originates from trans-splicing. Resistance to RNase R was used to rule out this possibility. (b) Results of RT-PCR. Each small box represents a PCR test and they were stacked. Five tests were performed for each type of transcript tested, on a mix of cDNAs and without/with pre-treatment of RNAs by RNAse R before the reverse transcription of RNAs from animals A-31 and A-65. When an amplicon of the expected size was observed, the box is colored black. Each negative result (white boxes) observed on cDNAs obtained after RNase R pre-treatment was confirmed by a second test (data not shown). When only a reduction in amplification was observed, the box is colored grey. In the second experiment (framed in dotted lines) concerning the RNase R resistance test of IntroLcirc-19, −116, −61,-68, −103, RT-PCRs were performed in parallel with two Taq polymerases. When positive amplification was observed with only one, the box is colored grey. (c) Evaluation of the level of ExoCirc-9244 by qRT-PCR. All 42 samples considered were previously tested for ExoCirc-9244 by RT-PCR.