Human Organ Tissue Identification by Targeted RNA Deep Sequencing to Aid in the Investigation of Shooting and Other Traumatic Bodily Injury Incidents

As submitted by the proposer: Molecular analysis of the RNA transcriptome from a putative tissue fragment should permit assignment of its source to a specific organ since each will exhibit a unique pattern of gene expression. Determination of the organ source of tissues from crime scenes may aid in shooting and other investigations. In shootings involving multiple bullets, guns and victims/participants, bullets and bullet fragments are recovered. If a recovered bullet possesses human tissue it might be possible to determine its precise trajectory through the human body. One bullet may contain brain tissue, another may contain a lung/skeletal muscle mixture and a third may contain adipose, thus indicating different trajectories and potentially different health outcomes. In some instances a crime is suspected of taking place at a particular location (e.g. in the home of an ex-wife who has disappeared) without a body being recovered. Traces of tissue at the scene (e.g. on the ceiling or wall) or on a suspect’s clothing may be found. If the tissue is identified as brain, as opposed to blood, and DNA testing indicated it was from the missing person, this would be an important piece of evidence indicating the likelihood of a fatal traumatic event. We have developed a prototype next generation sequencing (NGS) mRNA profiling assay for organ tissue identification designed to definitively identify ten organ/tissue types using a targeted panel of 46 mRNA biomarkers. The identifiable organs and tissues include brain, lung, liver, heart, kidney, intestine, stomach, skeletal muscle, adipose and trachea. The biomarkers were chosen after iterative specificity testing of numerous candidate genes in various tissue types. The assay is very specific with little cross reactivity with non-targeted tissue and can detect RNA mixtures from different tissues. Preliminary work has been conducted using preisolated tissue total RNA from multiple commercial sources, so further optimization of the assay is proposed here with the following studies concentrating on real tissue samples: finalize biomarker selection (e.g. additional adipose, trachea and spinal cord markers); test tissue samples from commercial sources; evaluate and refine the limit of detection (LOD), sensitivity and specificity. Prior to implementation in casework, further evaluation of the assay using post mortem tissue is essential. This includes determination of the sensitivity and specificity in freshly autopsied tissue samples and subsequently on the same autopsied tissues dried onto substrates including bullets and knives. Once the latter study is carried out technology transfer to interested laboratories can take place. Note: This project contains a research and/or development component, as defined in applicable law. ca/ncf