Body fluid identification is critical for forensic investigations, providing essential information for crime scene reconstruction. Common biomarkers for body fluid identification, such as DNA methylation, mRNA, and miRNA, have been widely applied. Recently a novel class of biomarkers, tRNA-derived small RNAs (tsRNAs), has been applied in some clinical studies and has demonstrated body fluid specificity. In our preliminary work, two epithelial-derived body fluids were used as a starting point to explore the potential of tsRNAs for body fluid identification, where tsRNAs yielded more differentially expressed candidates than miRNAs. Therefore, we aim to systematically evaluate both miRNA and tsRNA expression profiles for the identification of five common body fluids: venous blood, semen, vaginal fluid, menstrual blood, and saliva in this study. Based on the Arraystar Human Small RNA Microarray data, candidate markers were screened using a stepwise strategy: (1) top 10 candidates were initial selected ranked by FC across pairwise comparisons among the five body fluids; (2) removal of non-specific markers expressed across multiple fluids from step (1); (3) exclusion of low-abundance targets (signal intensity < 10); and (4) prioritization of the remaining markers showing consistent body fluid–specificity across multiple comparisons through. In addition, we applied two complementary strategies were further incorporated to identify more body fluid–specific markers: group-based comparisons (blood vs. non-blood; epithelial vs. non-epithelial origin) and an integrated ranking approach combining expression level, statistical significance, and FC. Selected markers were validated by RT-qPCR with stem-loop reverse transcription using U6 snRNA as an internal reference. The results showed that identified differentially expressed tsRNAs yielded a greater overall amount (4254) than miRNAs (2626) through pairwise comparisons across most body fluids, except in semen. Notably, three vaginal fluid–specific tsRNA markers identified in this study were also observed in our previous study, supporting their reproducible specificity for vaginal fluid. Finally, we developed a multiplex system including twelve tsRNA markers for the identification of five body fluids.

Authors

• Shengqiu Qu (Department of Forensic Gentics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Tangleixin Li (Department of Forensic Gentics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Zhilong Li (Laboratory of Molecular Translational Medicine, West China Second University Hospital, Sichuan University, China)
• Yuanyuan Xiao (Department of Forensic Gentics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Weibo Liang (Department of Forensic Gentics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)