Wang Dan-Yang, Tian Mei-Hui, Chen Yun-Zhou, Wang Si-Wen, Xing Xin-Yu, Sun Mao-Ling, Liu Zhenze, Liu Yalin, Wang Hongbo, Wei Jiayi, Zhong Yang, Yao Jun
School of Forensic Medicine, China Medical University, Shenyang 110000, PR China; Key Laboratory of Forensic Bio-evidence Sciences, Shenyang, Liaoning Province 110000, PR China; China Medical University Center of Forensic Investigation, Shenyang 110000, PR China.
Cardiac Intensive Care Unit, Shenyang Medical College Affiliated Second Hospital, Shenyang, PR China.
Forensic Sci Int Genet. 2025 Mar;76:103230. doi: 10.1016/j.fsigen.2025.103230. Epub 2025 Jan 25.
It is challenging to distinguish monozygotic (MZ) twins using traditional autosomal STR genotyping due to their nearly identical genomes. As an important kind of small non-coding RNAs, microRNAs (miRNAs) are essential regulators of gene expression and considered as excellent biomarkers due to their resistance to degradation. Moreover, droplet digital PCR (ddPCR) has emerged as a powerful technique for detecting gene mutations and pathogenic microorganisms, owing to its sensitivity and reliability. We aimed to explore the differential expression of miRNAs between MZ twins using next-sequence platform and assess the reliability of differentially expressed miRNAs by ddPCR. MiRNA sequencing (miRNA-seq) revealed nine differentially expressed miRNAs shared across five pairs of twins, including hsa-miR-3620-3p, hsa-miR-6825-5p, hsa-miR-1273h-5p, hsa-miR-200a-5p, hsa-miR-3192-5p, hsa-miR-188-5p, hsa-miR-206, hsa-miR-4796-5p, and hsa-miR-6775-3p. Subsequently, the combination of real-time quantitative PCR (qPCR) and ddPCR confirmed the ability of five of these miRNAs (hsa-miR-1273h-5p, hsa-miR-3192-5p, hsa-miR-188-5p, hsa-miR-206, and hsa-miR-6775-3p) in distinguishing monozygotic twins. Furthermore, ddPCR demonstrated superior recognition accuracy compared to qPCR. Finally, we evaluated the degradation resistance of these five miRNAs under different environmental conditions. None of the five miRNAs showed a significant decrease in expression levels after being stored at room temperature for up to 180 days or undergoing 10 freeze-thaw cycles. In summary, our study revealed the potential application of miRNAs in differentiation of MZ twins and the powerful role of ddPCR in forensic medicine.
由于单卵双胞胎（MZ）的基因组几乎完全相同，使用传统的常染色体短串联重复序列（STR）基因分型来区分他们具有挑战性。作为一类重要的小非编码RNA，微小RNA（miRNA）是基因表达的重要调节因子，因其抗降解性而被视为优秀的生物标志物。此外，数字液滴PCR（ddPCR）因其灵敏度和可靠性，已成为检测基因突变和致病微生物的强大技术。我们旨在使用下一代测序平台探索MZ双胞胎之间miRNA的差异表达，并通过ddPCR评估差异表达miRNA的可靠性。miRNA测序（miRNA-seq）揭示了五对双胞胎中共同存在的9种差异表达的miRNA，包括hsa-miR-3620-3p、hsa-miR-6825-5p、hsa-miR-1273h-5p、hsa-miR-200a-5p、hsa-miR-3192-5p、hsa-miR-188-5p、hsa-miR-206、hsa-miR-4796-5p和hsa-miR-6775-3p。随后，实时定量PCR（qPCR）和ddPCR的联合应用证实了其中5种miRNA（hsa-miR-1273h-5p、hsa-miR-3192-5p、hsa-miR-188-5p、hsa-miR-206和hsa-miR-6775-3p）具有区分单卵双胞胎的能力。此外，与qPCR相比，ddPCR显示出更高的识别准确性。最后，我们评估了这5种miRNA在不同环境条件下的抗降解能力。在室温下储存长达180天或经历10次冻融循环后，这5种miRNA的表达水平均未出现显著下降。总之，我们的研究揭示了miRNA在区分MZ双胞胎方面的潜在应用以及ddPCR在法医学中的强大作用。
