Differentiation of five forensically relevant body fluids using a small set of microRNA markers

In forensic investigations, identifying the type of body fluid allows for the interpretation of biological evidence at the activity level. Over the past two decades, significant research efforts have focused on developing molecular methods for this purpose. MicroRNAs (miRNAs) hold great promise due to their tissue-specific expression, abundance, lack of splice variants, and relative stability. Although initial findings are promising, achieving consistent results across studies is still challenging, underscoring the necessity for both original and replication studies. To address this, we selected 18 miRNA candidates and tested them on 6 body fluids commonly encountered in forensic cases: peripheral blood, menstrual blood, saliva, semen, vaginal secretion, and skin. Using reverse transcription quantitative PCR analysis, we confirmed eight miRNA candidates (miR-144-3p, miR-451a, miR-205-5p, miR-214-3p, miR-888-5p, miR-891a-5p, miR-193b-3p, miR-1260b) with high tissue specificity and four (miR-203a-3p, miR-141-3p, miR-200b-3p, miR-4286) with lesser discrimination ability but still contributing to body fluid differentiation. Through principal component analysis and hierarchical clustering, the set of 12 miRNAs successfully distinguished all body fluids, including the challenging discrimination of blood from menstrual blood and saliva from vaginal secretion. In conclusion, our results provide additional data supporting the use of a small set of miRNAs for predicting common body fluids in forensic contexts. Large population data need to be gathered to develop a body fluid prediction model and assess its accuracy.