Evaluating stability of miR-891a and miR-10a markers in semen samples to various conditions

MicroRNAs (miRNAs) have emerged as promising biomarkers for body fluid identification in forensic biology because of their small size and tissue-specific patterns. Their relative stability compared with other RNA molecules makes them potential alternatives in cases where DNA-based analysis is limited by degradation or insufficient sample quantity. The aim of this study is to evaluate the stability of miR-10a and miR-891a in semen samples exposed to environmental conditions relevant to forensic casework. Semen samples obtained from five healthy male volunteers were deposited onto 100% cotton fabric and exposed to four environmental conditions: room temperature, burial in soil, submersion in lake water, and washing at 90°C with detergent. Total RNA was isolated using a spin-column-based extraction kit, followed by cDNA synthesis and quantitative real-time PCR (RT-qPCR) analysis. Relative expression levels were normalized using RNU6b as a reference gene and evaluated using the 2−ΔΔCt method. miR-891a showed greater relative stability than miR-10a under the tested conditions. Increased miRNA degradation was observed in samples exposed to high temperature and detergent, and miRNA recovery was not achieved in these samples was not possible. Although condition-related differences were observed, statistical analysis did not show significant overall effect of environmental exposure on miRNA stability (p > 0.05).  miR-891a may represent a promising biomarker for semen identification in forensic casework. Nevertheless, the results should be interpreted cautiously, as recovery was limited under extreme exposure conditions. Additional studies with larger sample sizes are required to further evaluate the applicability of these markers in degraded samples.