Temperature-dependent stability of the miR-451a/miR-21-5p expression ratio for forensic discrimination of menstrual from peripheral blood.

BACKGROUND: Distinguishing peripheral blood (PB) from menstrual blood (MB) is critical in forensic body fluid identification, particularly in sexual assault and violent crime investigations. The miR-451a/miR-21-5p ratio has been proposed as a discriminatory marker, but its stability under environmentally relevant temperatures has not been systematically evaluated. AIM: To assess the effect of three temperatures (4 °C, 24 °C, and 40 °C) on the discriminatory performance of the miR-451a/miR-21-5p ratio for PB-MB differentiation, and to compare ratio-based performance against individual miRNA analysis. METHODS: Forty blood samples (20 PB, 20 MB) were collected from healthy female volunteers. Total RNA was extracted, and miR-451a and miR-21-5p levels were quantified by RT-qPCR after 24 h of exposure to each temperature. RESULTS: The ratio was significantly higher in PB than in MB at all temperatures (P < 0.001). Individual miRNA analysis was less reliable: miR-451a significantly differentiated PB from MB only at 4 °C and 40 °C, while miR-21-5p did not differentiate between blood types at any temperature. Receiver operating characteristic (ROC) analysis identified cut-offs of 1.06, 1.02, and 0.765 at 4 °C, 24 °C, and 40 °C (all P < 0.001), with sensitivity/specificity of 70/90%, 100/100%, and 100/100%, respectively. A universal, temperature-independent cut-off (<0.900) also achieved acceptable discrimination (AUC 0.927; sensitivity 96.7%, specificity 86.7%). CONCLUSION: The miR-451a/miR-21-5p ratio maintained discriminatory performance across all temperatures and outperformed individual miRNA markers. However, the observed differences reflect the combined effect of temperature and storage format rather than temperature alone; therefore, future studies under identical dried conditions are needed to isolate the temperature effect.