A Rapid, Microfluidic Transcriptomic Method for Body Fluid Identification from Forensic Samples

Forensic science employs techniques in genomics, transcriptomics, and proteomics that stem from a solid molecular biology foundation. Unknown stains at a crime scene undergo proteomic-based serology to decipher the tissue of origin. This critical information helps investigators corroborate a witness or form hypotheses, and potentially identify suspects based on genomic makeup. While DNA analysis is unwavering in its role in forensic science, and proteomics has been prevalent, we are experiencing a paradigm shift towards transcriptomics to identify the origin of unknown stains, enabling an improved evidence-informed sample selection process for subsequent DNA analysis. In addition to high sensitivity and specificity in transcriptomic (mRNA-based) assays, sample preparation can allow for the simultaneous isolation of RNA during DNA extraction. Consequently, evidence consumption can be minimized while presenting the possibility for mRNA and DNA to be analyzed in parallel. In previous NIJ-funded work, we demonstrated mRNA targets for the identification of venous blood, menstrual blood, semen, saliva, and vaginal fluid. In addition, human male DNA was included to assist in identifying probative sexual assault samples. Here, we propose leveraging those developments, but using the mRNA targets defined by ESR shown to provide unparalleled specificity and, with fluorescence detection, superb sensitivity. While the current read-out for the ESR panel is size-based electrophoretic separation on ABI 3500, the sample preparation remains burdensome and time-consuming. We propose addressing this problem with a microfluidic approach that expedites and automates the multistep process, and opens up new opportunities for improved throughput. Sample insertion, RNA extraction and real-time polymerase chain reaction will be carried out on a microfluidic disc where fluid flow, centrifugally driven, allows for a series of sequential steps (mixing, aliquoting, heating, storage) to execute an automated sample preparation. The result of the on-disc processing with on-disc reagent will be amplified fragments that, through capillary electrophoretic separation, define the presence or absence of five body fluids (while retaining extracted DNA for other analyses). In parallel, we will further the work we have done establishing a disc-based electrophoresis system for ultrafast DNA fragment separation. In summary, the overall goal of this proposal is to generate prototypes for body fluid identification that can be trialed in two forensic laboratories one at ESR in New Zealand and the other at a forensic laboratory in the USA. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF