title: Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.
abstract: 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 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method 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 samples of blood, semen, vaginal secretions, menstrual blood and skin 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. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. 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.
authors.affiliation: NanoString Technologies, 530 Fairview Avenue N, Suite 2000, Seattle, WA 98109, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Danaher
authors.affiliation: NanoString Technologies, 530 Fairview Avenue N, Suite 2000, Seattle, WA 98109, USA.
authors.forename: Robin Lynn
authors.initials: RL
authors.lastname: White
authors.affiliation: National Center for Forensic Science, P.O. Box 162367, Orlando, FL 32816-2367, USA.
authors.forename: Erin K
authors.initials: EK
authors.lastname: Hanson
authors.affiliation: National Center for Forensic Science, P.O. Box 162367, Orlando, FL 32816-2367, USA; Department of Chemistry, University of Central Florida, P.O. Box 162366, Orlando, FL 32816-2366, USA. Electronic address: Jack.Ballantyne@ucf.edu.
authors.forename: Jack
authors.initials: J
authors.lastname: Ballantyne
mesh_terms: D001331:Automation; D001826:Body Fluids / Q000378:metabolism*; D053803:Forensic Genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger
keywords: Body fluid identification; De-convolution of body fluid mixtures; Forensic science; Gene expression; NanoString(®); mRNA profiling