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

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.

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.

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.

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%).

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.