Human Organ Tissue Identification by Targeted RNA Deep Sequencing to Aid the Investigation of Traumatic Injury

Abstract

Molecular analysis of the RNA transcriptome from a putative tissue fragment should permit the 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 shootings and other investigations. We have developed a prototype massively parallel sequencing (MPS) mRNA profiling assay for organ tissue identification that is designed to definitively identify 10 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. We also demonstrate the ability of the assay to successful identify the tissue source of origin using a single blind study.

Identification of cadaveric liver tissues using thanatotranscriptome biomarkers

“…The primary aim of the current study is to validate the use of an MPS-based organ tissue identification assay at varying times of death using actual criminal casework. The study builds upon previous findings of the thanatotranscriptome that described targeted RNA expression analyses by ten commercially acquired organ tissue types using a targeted panel of 46 mRNA biomarkers 5 . This assay precisely identified tissue source origins using a single blind study of RNA mixtures from different internal organs.…”

“…Emerging research to clarify the postmortem fingerprint of mRNA transcript expression patterns of the 25,000 human genes 1,2 have been studied to clarify the secrets of how human beings die. Integrated approaches to investigate temporal variations and the eventual cessation of postmortem transcription expression patterns have influenced an upsurge in studies of the functional complexities of the thanatotranscriptome [3][4][5][6][7][8] . The thanatotranscriptome is derived from the Greek word for death (thanatos-), and it encompasses all RNA transcripts expressed from the part of genome that is still functional or that becomes awakened in internal organs of a dead body 3 .…”

“…Previous studies have been performed using mRNA-based approaches to supplement traditional protein-based methods of identifying organ tissue sources 5,8 . The integrity of postmortem tissues samples is of paramount importance in regard to experiments that attempt to monitor gene expression at the precise stage of RNA extraction.…”

“…www.nature.com/scientificreports www.nature.com/scientificreports/ TruSeq targeted RNA library preparation. As previously described, a targeted RNA deep sequencing that included a massively parallel sequencing (MPS) multiplex of 46 genes was employed 5 . Briefly, custom sequencing panels were prepared using the TruSeq Targeted RNA Expression kit (Illumina, Inc.), and the Illumina DesignStudio was used to design a custom TruSeq Targeted oligonucleotide pool (TOP).…”

“…TruSeq Targeted RNA custom TOP library preparation. cDNA was hybridized to the custom TOP as previously described 5 . Briefly, the 10 µL reaction mixture contained 5 µL of the oligonucleotide pool (Illumina Inc.) and 5 µL of Tris-ETDA buffer (pH 8.0) (Thermo Fisher Scientific).…”