CircRNA profiles from one-tube multiplex probe-based qPCR can distinguish between monozygotic twins

Monozygotic twins (MZTs) pose a considerable challenge in personal identification due to their nearly identical genomic DNA sequences. Emerging evidence suggests that epigenetic markers, particularly non-coding RNAs (ncRNAs), may provide a promising strategy to discriminate MZTs. Circular RNAs (circRNAs) feature high stability and tissue-specific expression and have shown considerable potential in forensic applications such as body fluid identification and age estimation. In this study, we performed whole-transcriptome circRNA sequencing on peripheral blood samples from five MZT pairs to screen for differentially expressed circRNAs (DEcircRNAs). A total of 13,191 circRNAs were identified, and six DEcircRNAs (hsa_circ_0018054, hsa_circ_0003413, hsa_circ_0067717, hsa_circ_0003941, hsa_circ_0107772, and hsa_circ_0058520) were confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) in an expanded cohort of 12 MZT pairs. Furthermore, these markers exhibited high stability in dried bloodstains stored for up to 180 days. Additionally, we developed a novel one-tube multiplex probe-based qPCR system for simultaneous detection of three circRNAs (hsa_circ_0003413, hsa_circ_0067717, and hsa_circ_0058520). This system enabled sensitive detection with a minimum requirement of just 4 ng of total RNA. Collectively, our study demonstrates that circRNAs are differentially expressed in MZTs, representing a novel epigenetic biomarker set for forensic individual identification. Our findings support the potential of circRNAs as stable and informative biomarkers to discriminate monozygotic twins in forensic practice.