Differential expression of long non-coding RNAs can effectively distinguish monozygotic twins.

Due to the nearly identical genomic sequences of monozygotic twins (MZTs), traditional autosomal Short Tandem Repeat (STR) genotyping methods are unable to distinguish between them. Epigenetic factors exhibit potential value in forensic science. Dynamic epigenetic factors may be influenced by genetic traits or postnatal environmental factors. This study focused on long non-coding RNAs (lncRNAs), and aimed to comprehensively screen for differentially expressed lncRNAs between MZTs using next-generation sequencing (NGS). Additionally, the consistency of these differentially expressed lncRNAs across multiple twin pairs was evaluated by quantitative PCR (qPCR). Through lncRNA sequencing (lncRNA-seq), a total of 12,547 expressed lncRNAs were identified in four pairs of MZTs, revealing 31 commonly differentially expressed lncRNAs. In the PCR preliminary experiment, 11 lncRNAs showed stable amplification effects and high specificity, providing a basis for further validation, including MSTRG.30665.2, ENST00000414030, MSTRG.73422.1, MSTRG.87582.24, MSTRG.100816.5, MSTRG.54028.15, MSTRG.16169.7, MSTRG.98182.1, MSTRG.68271.29, MSTRG.63897.85, and MSTRG.16294.1. Subsequently, qPCR confirmed the identification ability of these lncRNAs. The identification ability of these 11 lncRNAs varies, with MSTRG.73422.1 having the strongest discrimination power. The forensic applicability assessment showed that five lncRNAs exhibited strong resistance to degradation under specific environmental conditions and robust longitudinal stability. Their expression levels did not significantly decrease after storage at room temperature for up to 180 days or following 10 freeze-thaw cycles. In summary, our study reveals the potential application of lncRNAs in the identification of MZTs.