The Potential Use of Tissue-Specific MicroRNAs for Forensic Sample Identification,

Background: In forensic casework, the identification of human material, especially at the scene can be extremely important. Several methods exist to date, but sometimes they lack sensitivity and specificity at detecting biological stains from human sources. Forensic RNA research using mRNA markers is still in its infancy but has provided the possibility of human stain identification. However, the use of miRNAs has not been recently proposed. Aims: The study was designed to cover four main issues: to differentiate between different tissue-types such as blood and saliva using miRNA specific-expressive markers; investigate the sensitivity of miRNAs; miRNA stability in forensic stains stored at various time and temperature conditions and obtaining successful DNA and miRNA profiling from a single bloodstain. DNA interference on miRNA targeting of TaqMan® primers and probes was also investigated. The main aim of the study was to provide evidence of miRNA-specific expression for biological stain (mainly blood) and tissue typing. Methods: MiRNA (absolute) quantification using qRT-PCR using miRNA TaqManTM assays were performed for miRNA profiling. DNA/RNA co-extractions were used, with PCR and Capillary Electrophoresis analyse used to obtain DNA profiles. Results: MiR-451 and miR-1 were detected in high abundance from 50μl dried blood stains and ~9mg skeletal muscle respectively, with little detection amongst other tissues types. Leukocytic-specific miRNAs (miR-150, -155, -223) possessed high cross-reactivity with saliva stains and were not found to be specific to blood. All miRNA primer and probe sets showed high sensitivity (average 9 logs) at detecting synthetic oligoribonucleotides. MiRNA stability during temperature and time storage was high, although variations were observed due to insufficient extraction method. Conclusions: MiRNAs, especially miR-451 is useful and could be used as a bloodstain marker. MiRNA could be used as an alternative to reported mRNA tissue profiling and current immunological and biochemical methods. Obtaining more miRNA markers and further validation was is required.