Biological traces recovered from crime scenes are often present in trace amounts and constitute important forensic evidence for reconstructing events. Estimating the time since deposition (TsD) of such evidences can contribute to spatiotemporal interpretation and support investigative decision-making. However, the applicability of current TsD inference methods to trace biological samples remains limited due to insufficient sensitivity and specificity, etc. As a result, there is a need to explore alternative molecular markers with improved detectability and temporal responsiveness for TsD estimation in trace biological evidence. This study aims to evaluate the feasibility of mitochondrial RNA (mtRNA) as a molecular marker for TsD estimation in trace biological samples. Specifically, we seek to characterize the temporal degradation patterns of selected mtRNA markers within the first three days after deposition and to assess their potential as sensitive and time-resolved indicators for TsD inference. Peripheral blood samples were collected from ten volunteers with informed consent and stored under ambient conditions to simulate post-deposition environments. Three mtRNA markers were selected for analysis, including 12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I (COI). Samples were obtained at three post-deposition time points: day 1, day 2, and day 3. Total RNA was extracted and quantified using qPCR method, with each marker measured in triplicate for each sample. Melting curve analysis was performed to assess amplification specificity, and relative RNA abundance was evaluated based on Ct values across different time points. All three mtRNA markers (12S rRNA, 16S rRNA, and COI) exhibited single-peak melting curves, confirming the specificity of primer amplification. Quantitative analysis demonstrated a progressive increase in Ct values over time, indicating a corresponding decrease in mtRNA abundance following deposition. Notably, 12S rRNA and 16S rRNA displayed relatively higher abundance levels, indicating their potential suitability as sensitive molecular targets for detection and application in trace biological evidence. These findings suggest that mtRNA exhibits measurable, time-dependent degradation within the early post-deposition period and may serve as a feasible marker for TsD estimation in trace biological samples. To enhance reliability, future studies should incorporate appropriate reference genes for normalization, expand sample size, validate findings across multiple platforms, and evaluate the influence of environmental factors on mtRNA degradation dynamics.

Authors

• Chong Chen (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)
• Jing Wang (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)
• Ying Zhu (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)
• Minzhu Zhao (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)
• Yongguo Li (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)
• Renkuan Tang (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)
• Jianbo Li (Department of Forensic Medicine, Faculty of Basic Medical Science, Chongqing Medical University, China)