Quantitative differential analysis of tsRNAs for forensic body fluid identification: RT-qPCR-based discrimination derived from epithelial cell fluids screening

Identification of body fluid types at crime scenes is a critical step in forensic science, providing essential context for criminal investigations and the interpretation of evidence. Epigenetic markers, particularly small non-coding RNAs (sncRNAs), have attracted increasing attention in forensic body fluid identification, with various small RNA species demonstrating potential as biomarkers for distinguishing different body fluid types. A novel class of sncRNAs, tRNA-derived small RNAs (tsRNAs), has been detected in various biological samples, yet their potential application in forensic body fluid identification remains unexplored. In this study, we identified differentially expressed tsRNAs in saliva and vaginal secretions, two epithelial cell-derived body fluids. Using stem-loop reverse transcription followed by SYBR Green quantitative polymerase chain reaction (RT-qPCR), we measured tsRNA abundance with U6-snRNA as the reference control. After implementing quality control measures to ensure detection accuracy and adjusting Cq values, we identified 10 tsRNAs that were differentially expressed between saliva and vaginal secretions. To further investigate the potential of tsRNAs in forensic body fluid identification, we extended our analysis to include semen, peripheral blood, and menstrual blood. Our results revealed 18 tsRNAs with significant differential expression patterns across these five body fluids. By integrating quantitative expression data with a logistic regression machine learning model, a panel of 9 tsRNAs achieved 98 % accuracy in leave-one-out cross-validation. While our findings demonstrate the promising potential of tsRNAs as biomarkers for forensic body fluid identification, additional body fluid-specific tsRNAs would likely enhance prediction accuracy while potentially reducing the number of required markers. This preliminary validation establishes tsRNAs as valuable candidates for forensic body fluid identification applications.