Ribonucleic Acid Fluorescence in situ Hybridisation for Forensic Cell Identification and Separation

Abstract

The identification of body fluids associated with crime is a critical part of forensic investigation. Accurate, specific and sensitive testing is required and genetic techniques have come to the forefront enhancing the abilities of investigators to analyse evidence. When crimes occur, the nature of biological interaction means that mixed samples of biological material can be generated, but interpretation of genetic results becomes complex when mixtures of fluids and donors are present. This work aimed to address this issue, considering samples at the single cell level, using fluorescence in situ hybridisation (FISH). Methods of labelling forensically relevant buccal, vaginal, blood and seminal cells were developed using both slide and suspension FISH methodologies coupled with locked nucleic acid probes, a technique which has not previously been proven effective with forensic samples. A range of mRNA and miRNA markers were screened for specificity with the developed protocol, focusing on differentiation between morphologically identical buccal and vaginal cells, but no markers were identified as showing specific fluorescent signal to either cell type. This reflects the difficulties present in the search for specificity due to gene expression similarities between these cell types, with the difference between these and published PCR based results indicative of the issues which arise when different platforms are used to generate gene expression data. Addressing the mixture issue in forensic interpretation, the culmination of this project was considering the isolation of these labelled cells and subsequent DNA profiling. Laser microdissection (LMD) and fluorescence activated cell sorting (FACS) were both successfully used to separate RNA FISH labelled epithelial cells with DNA profiling possible from these cell isolates. Furthermore, FACS was able to separate mock mixture RNA-FISH labelled samples, showing minimal cross contamination. Each technique showed promise for forensic application but were more suited to different sample types; LMD for restricted cell numbers and FACS for abundant cell numbers. This proof-of-concept study, the first of its kind in the forensic arena, shows that RNA FISH coupled with LMD or FACS is a valuable tool for resolving forensic mixtures.