A new strategy for distinguishing menstrual blood from peripheral blood by the miR-451a/miR-21-5p ratio.

Distinction between menstrual blood and peripheral blood is vital for forensic casework, as it could provide strong evidence to figure out the nature of some criminal cases. However, to date no single blood-specific gene, including the most variable microRNAs (miRNAs) could work well in identification of blood source. In this study, we developed a new strategy for identification of human blood samples by using the copy number ratios of miR-451a to miR-21-5p based on 133 samples, including 56 menstrual blood and 47 peripheral blood, as well as 30 non-blood samples of saliva (10), semen (10) and vaginal secretion (10). The cut-off value and efficacy of the identification strategy were determined through receiver operating characteristic (ROC) analysis. Our results showed that when the miR-451a/miR-21-5p ratio below 0.929, the sample should be non-blood. In contrast, when the miR-451a/miR-21-5p ratio above 0.929 and below 10.201, the sample should be menstrual blood; and when this ratio above 10.201, the sample should be peripheral blood. External validation using 86 samples (62 menstrual blood and 24 peripheral blood samples) fully supported this strategy with the 100% sensitivity and 100% specificity. We confirmed that this result accuracy was not affected by various potential confounding factors of samples and different experimental platforms. We showed that 0.2 ng of total RNA from menstrual blood and peripheral blood was sufficient for qPCR quantification. In conclusion, our results provide an accurate reference to distinguish menstrual blood from peripheral blood for forensic authentication.