RNA-based identification of forensically relevant body fluids/tissues – novel strategies and methods for casework implementation

Abstract

This dissertation includes several objectives that will lead to an improvement in the identification of forensically relevant body fluids/tissues using RNA in the context of casework. The determination of body fluids/tissues is of great importance in forensics to reconstruct the circumstances of a crime. In this way, important aspects can be identified that are of great relevance for a verdict in court. Various research approaches, methods, and implementations for forensic casework have been explored to improve the validity and spectrum of body fluid identification. In the first part, an mRNA assay for body fluid/tissue identification was adapted and validated to obtain a highly sensitive method. Comparison of different DNA/RNA co-extraction and re- extraction approaches using an automated extraction instrument showed how effectively re- extraction can be used to obtain trace-preserving results for DNA and RNA typing. This DNA/RNA re-extraction was tested on a variety of casework samples. It was found that successful determinations of body fluids were obtained from the already DNA-extracted swabs in a large proportion of the casework samples. Therefore, this approach can be well used for casework as there is no loss of DNA yield. Examination of the forensically relevant body fluid rectal mucosa has been neglected in research, although the number of sexual offenses involving anal penetration is large. For this reason, an innovative marker for the rectal mucosa was developed and successfully integrated to the mRNA assay. The validation of this assay already showed new insights into the specificity of the established markers and weaknesses as well as strengths of the assay. This mRNA assay was further applied in a transfer study investigating the persistence of blood on shoe soles. The transfer study included DNA typing, determination of body fluids using mRNA, and comparison with other established determination methods. These results provide important insights for the determination of blood traces as well as the persistence of blood and the extrapolation to the activity level that can be derived from this. In addition, a DNA/RNA co-extraction was developed for this study, which is specifically appropriate for the examination of micro traces. The last study involves an approach to simultaneously investigate mRNA and miRNA markers. This multiplex assay can combine the advantages of both types of RNA. There is a plethora of specific and sensitive mRNA markers, and miRNA markers exhibit high resistance to degradation. Initial results demonstrate the advantage of this novel combination for the study of old, degraded samples. For the first time, an mRNA/miRNA multiplex assay with capillary electrophoretic analysis was successfully tested and established. This innovative approach can also be easily applied to research questions in other molecular fields and can be implemented in casework. The results of this work provide important new approaches and insights in the field of body fluid/tissue determination that can further advance this area of research. The dissertation further demonstrates what has to be considered when implementing these assays in casework and points out the limitations as well as new possible approaches.