Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets

A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString® probes to 23 tissue/body fluid specific mRNA targets. The body fluids/tissues targeted were peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We tested and compared a simple 5 minute room temperature cellular lysis protocol against standard RNA isolation from same source material as a means to facilitate ease-of-use in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source blood, semen, vaginal secretions, menstrual blood and skin samples all demonstrated the expected tissue specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. Two of the five mixtures were called perfectly using the assay algorithm, and one of the component fluids was identified in the each of the 'false negative' mixtures. Crucially, our algorithm produced zero false positive fluid identifications across this study's 98 samples. Further optimization of the biomarker 'Codeset' will be required before it can be used in casework, particularly with respect to increasing the signal to noise ratio of the saliva biomarkers. With suitable modifications, this simplified protocol with minimal hands on requirement should facilitate routine use of mRNA profiling in casework laboratories.