MicroRNA profiling for the identification of forensically significant body fluids in mixed samples

<p dir="ltr">In forensic science, ‘body fluids’ refers to biological stains recovered from surfaces outside the originating body. Their identification is critical for crime scene reconstruction and determining their origin. However, conventional body fluid identification (BFID) methods, such as serological and immunological assays, lack specificity and sensitivity, particularly in cases involving unknown or mixed body fluids.</p><p dir="ltr">MicroRNAs (miRNAs), a class of small non-coding RNAs, typically 18-24 nucleotides long, have emerged as promising biomarkers for BFID owing to their tissue-specific expression and resistance to environmental degradation. While previous studies have explored miRNA expression in individual body fluids, research on their application in mixed samples remains limited.</p><p dir="ltr">This study aimed to develop a miRNA-based assay for the identification of forensically significant body fluids in mixed samples. A systematic review following PRISMA guidelines identified body fluid-specific miRNAs, and nucleic acid isolation methods and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols were optimised and validated for this purpose. A proof-of-concept study evaluated the efficiency of different extraction kits for the co-isolation of DNA, RNA, and miRNA from human saliva, revealing significant variations in nucleic acid yield, quality, and miRNA expression, by kit used.</p><p dir="ltr">A panel of 14 miRNAs was screened for expression and stability in blood, buccal swab, menstrual blood, and vaginal secretion samples. From this, a discriminative panel (miR-126, miR-451a, miR-144-3p, and miR-203) was developed, successfully differentiating blood and vaginal secretion, and blood and buccal swab mixtures, using RT-qPCR.</p><p dir="ltr">Next-generation sequencing (NGS) identified 20 differentially expressed miRNAs in menstrual blood and vaginal secretions, including mir-224, mir-32, and mir-548ad, which are newly reported as potential candidates for forensic BFID. t-distributed stochastic neighbour embedding (t-SNE) visualisation revealed distinct clustering between these fluids, demonstrating the potential of NGS for complex BFID.</p><p dir="ltr">These findings lay the foundation for future research, focusing on the development of specific qPCR probes for the newly discovered miRNAs, thereby integrating the advantages of sequencing with targeted amplification techniques. This study highlights the potential of miRNA profiling as a powerful tool for forensic BFID, supporting its integration into forensic casework and advancing forensic molecular biology.</p>