Developing a microRNA body fluid identification test for use in forensic casework

Body fluid identification (BFID) forms a crucial aspect within forensic investigations. BFID is particularly important where the origin of a DNA profile is also needed e.g. sexual assaults. The aim of this work was to develop a RNA-based BFID for use in forensic casework. The test was developed in three stages: exploring co-isolation and single isolation strategies, screening and selecting RNA markers and different casework conditions e.g. low-level, non-human, degraded and mixed samples. Blood, saliva, skin, semen and vaginal material samples were collected. Samples underwent single or co-isolation (DNA, total RNA or messenger RNA), DNA quantification, cDNA synthesis and qPCR using a number of different candidate markers and reference genes for microRNA (miRNA) and messenger RNA. During the development of the test the following novel findings were observed: utilisation of complex co-isolation methods was not need for co-analysis of RNA and DNA; six highly discriminative miRNA markers were identified for miRNA analysis: blood (miR-451 and miR-194); saliva (miR-205); vaginal material (miR-224 and miR-335) and semen (miR-891a). In terms of casework applications, microRNA analysis showed potential for greater sensitivity than current enzymatic methods with the use of appropriate reference gene; species specificity was observed for reference gene RNU44; sample stability was observed in 1-yr bloodstains with miR-451 and RNU44 and successful resolution of major and minor components was achieved.