Development and Evaluation of miRNA and mRNA Panels for Body Fluid Identification

The attribution of biological material to a tissue source, also known as body fluid identification (BFID), can aid investigators in corroborating statements and in the reconstruction of events. Chemical tests, microscopy, enzymatic activity, and immunochromatographic tests are widely employed in crime laboratories at present but have a high false-positive rate and interpretations are often subject to conjecture. Forensic testing requires a methodology that can analyze low level and challenging samples with high specificity. While several molecular targets have been considered for BFID, this project explores the use of microRNA (miRNA) and messenger RNA (mRNA) for BFID. The results of this study provide the forensic community with information on the performance of miRNA and mRNA for BFID using capillary electrophoresis (CE), quantitative reverse transcription PCR (RT-qPCR), and massively parallel sequencing (MPS) technologies, particularly for challenging samples. In the first study, an eight-marker miRNA multiplex was developed for capillary electrophoresis using a linear primer system. Markers were chosen to identify venous blood, menstrual blood, semen, saliva, as well as an endogenous reference gene. Reverse transcription and PCR primers were developed for each marker, evaluated in singleplex, and then multiplexed. Each fluid were co-extracted (DNA/RNA) and amplified with STR and miRNA multiplexes, respectively. All DNA profiles were complete and miRNA profiles correctly identified the body fluid using a decision tree-based interpretation strategy. In the second study, the stability and persistence of miRNA and mRNA was evaluated in challenging samples. Custom reverse transcription quantitative PCR (RT-qPCR) assays were performed to detect the presence of miRNA and mRNA in samples. mRNA targets and miRNA targets were chosen for blood, semen, and an endogenous reference gene respectively. For the evaluation of these markers over time, blood and semen samples were placed in a glass enclosed area exposed to natural heat, humidity, and UV light as well as controlled conditions in a lab cabinet (room temperature, low humidity, and darkness) for up to six months. mRNA was undetected in experimental samples after 30 days, while control mRNA and all miRNA transcripts were detected for the duration of the experiment. A persistence study was also performed by laundering blood and semen stained swatches and either machine drying or allowing the samples to air dry. Blood specific markers were detected in all bloodstained samples, while semen specific markers were observed in all but one semen stained sample. Transfer of both miRNA and mRNA was observed by taking an unstained portion of the swatch. In the final study, an early access mRNA panel for BFID was evaluated. Samples of venous blood, menstrual blood, semen, saliva, and vaginal secretions were placed in a variety of challenging conditions including outside with and without exposure to direct precipitation, buried, on a decomposing cadaver, laundered, aged, and post-coital samples. Co-extracted DNA profiles were evaluated, and RNA was successfully extracted and typed in most samples. Reverse transcription negatives, total reads, and composition of reads attributed to body fluid specific markers were used to evaluate the performance of the panel. Based on the results of the study, potential areas for improvement were highlighted; however, the performance of the panel overall is encouraging.