Fig. 6

NCX1 is a highly expressed and conserved circular RNA. a qPCR agrees with sequencing estimates and shows that circular isoforms of NCX1 are induced in the fetal heart and during in vitro cardiomyocyte differentiation. Plotted values are ΔCt = Ct(ACTB) – Ct(target); error bars are standard error of the mean of technical replicates for fetal heart, and of biological triplicates for human ESC (hESC) to cardiomyocytes. b Our de novo sequencing algorithm predicted a minor circular isoform differing by a deletion of three nucleotides from the dominant circular isoform; it arises from use of a splice-acceptor just downstream of the annotated splice-acceptor. The minor circular isoform was confirmed by PCR and clone sequencing. In the diagram, exonic sequences from genome annotations are given in bold uppercase, and splice-signal dinucleotides are highlighted in red; the mouse and rat NCX1 sequences are shown in blue. In the rat, the NCX1 circular isoform was only detected with the aid of our de novo algorithm, as the circle junction does not coincide with the annotated start of the first exon. Notably, in the mouse the start of this exon is annotated as exactly where we see circular splicing in the rat and mouse