FIGURE 4.

Validation of circular RNA species and “virtual Northern.” (A) Design of Taqman assays using outward facing primers. (B) Validation of RNase R enrichment in seven novel backsplice junctions and one control circular RNA (ANRIL 14-5). Noncircular RNAs (TBP, GAPDH, and 18S) are depleted by RNase R treatment. (C) Ratio of expression of these same ecirc and control RNAs with cDNA synthesis using oligo dT primers vs. random hexamer. Note markedly decreased cDNA synthesis with oligo dT for ecircRNAs and 18S. (D) “Virtual northern” analysis of four backsplice species and their linear counterparts employing agarose gel size fractionation followed by qPCR for quantification of products in each fraction. The x-axis shows size fraction in order of decreasing size, and the y-axis indicates the calculated fraction of total species contained at that size range.