Figure 4. General biological properties of ovary-derived circRNAs. (A) Primer patterns of circRNAs and linear RNAs. The blocks represent exons. Red arrows represent divergent primers, blue arrows represent convergent primers, and the black vertical line represents the circRNA back-spliced junction. (B) CircRNAs were successfully amplified by divergent primers from cDNA but could not be amplified from gDNA. Linear RNAs were successfully amplified by convergent primers from both cDNA and gDNA. PCR products were examined by 2% agarose electrophoresis. The circCCSER2 and circATXN3 were used as experimental groups, and GAPDH was the control group. (C) Total RNA derived cDNA successfully amplified circRNAs, but this did not work for poly-A+ RNA derived cDNAs. Four circRNAs were examined, and their linear forms were used as controls. (D) CircRNAs were resistant to RNase R digestion, whereas their linear forms were sensitive to RNase R digestion. Ten circRNAs were examined, and six of the corresponding linear RNAs were used as controls. (E) Ovary-derived circRNAs can be stably expressed in human granulosa cells (hGCs) from follicular fluid in 24 h, whereas their linear forms are degraded rapidly. (F) A schematic diagram of the alternative splicing. PCR products for three pairs of primers were examined by 2% agarose electrophoresis (G) and Sanger sequencing (H). CircCSE1L was explored as an example. *, P < 0.05; **, P < 0.01; ***, P < 0.001.