Investigation of the semen-specificity of various miRNA markers

Advancements in forensic sciences have led to the development of diverse approaches for the analysis of evidence encountered at crime scenes. While DNA-based methods dominate for identification purposes, RNA-based approaches are more prominent in body fluid/tissue identification, a critical parameter for reconstructing crime scenes. A review of the literature indicates that the primary limitation of messenger RNA (mRNA)-based methods is their susceptibility to degradation. Conversely, microRNA (miRNA) molecules are more resistant to degradation factors, making them highly promising for forensic applications. Non-coding microRNAs (nc-miRNAs) are small RNA molecules, 18–22 nucleotides in length, playing significant roles in epigenetic mechanisms and regulating gene expression. They achieve gene expression regulation by forming complexes with various proteins and binding to the 3' UTR regions of mRNAs, thereby downregulating their expression. Considering the tissue/body fluid-specific expression profiles of mRNAs, it can be inferred that the miRNAs regulating them exhibit similar specificity. Among the most frequently encountered body fluids at crime scenes are blood, semen, and saliva. Literature highlights miRNAs such as miR-10a-3p, miR-10a-5p, miR-888-3p, miR-888-5p, and miR-891a-3p as prominent candidates for semen identification. In this study, miR-10a-3p, miR-10a-5p, miR-888-3p, miR-888-5p, and miR-891a-3p markers were analyzed in samples of venous blood (n=10), menstrual blood (n=10), vaginal secretions (n=10), saliva (n=10), and semen (n=10) collected from 50 volunteers. Based on the literature, U6 and SNORD48 were selected as reference genes. Preliminary studies revealed that U6 was inefficient (Ct>40), and therefore SNORD48 was utilized in subsequent analyses. Grouping body fluids into semen and non-semen categories, discriminant analyses showed that miR-10a-3p (92.0%), miR-10a-5p (94.0%), miR-888-3p (94.0%), miR-888-5p (100.0%), and miR-891a-3p (100.0%) could accurately distinguish semen samples. Statistical analyses identified the combination of miR-888-3p, miR-888-5p, and miR-891a-3p as the most effective for semen identification. When these three markers were used together, they achieved 100.0% accuracy in distinguishing semen from other body fluids.