Biofluid identification in mock sexual assault samples using a semi-automated, extraction-free microRNA gene expression profiling method
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Abstract
Biofluid identification plays a pivotal role in forensic case investigation, as it assists in crime scene reconstruction and provides guidance for subsequent analysis. Current forensic biofluid detection techniques, such as alternate light sources, catalytic color tests, and lateral flow immunochromatographic assays, exhibit limitations in terms of sensitivity and specificity, and most can only detect one biofluid at a time. Developing a multiplex and confirmatory assay will significantly benefit forensic biofluid identification, with nucleic acid-based assays emerging as the most promising candidates. Among various nucleic acids, microRNAs boast biofluid specificity and exceptional stability, rendering them an optimal choice for biofluid identification assay and the focal point of this study.
In this study, we investigated a semi-automated, extraction-free microRNA gene expression profiling method developed by HTG Molecular Diagnostics, Inc., namely HTG EdgeSeq microRNA Whole Transcriptome Assay, utilizing seven biofluid types frequently encountered in sexual assault cases, including peripheral blood, menstrual blood, saliva, vaginal fluid, and semen with varying sperm counts. The research focused on three aspects: the assay’s compatibility with whole blood samples and dried biofluid swab samples, its ability to identify and classify different biofluid types based on their microRNA expression patterns, and its potential to differentiate biofluids within mixtures.
Our results demonstrate that HTG EdgeSeq microRNA Whole Transcriptome Assay effectively identified several differentially expressed microRNAs in each biofluid. Examples that align with the existing literature include miR-451a and 486-5p for all kinds of blood, miR-185-5p for peripheral blood, and miR-888-5p and 891a-5p for semen. Previously unreported microRNAs with biofluid-specific expressions were also discovered, such as miR-4306 for peripheral blood and miR-184 for saliva. The scatter plots and heat maps generated from experimental data exhibit distinctive microRNA expression profiles for different biofluid types, potentially enabling their identification and classification. Furthermore, the analysis of mixtures reveals that differentially expressed microRNAs in mixtures corresponded to those detected in the single-source biofluids, signifying the assay’s capacity to resolve biofluid mixtures. The assay also yields optimal results for whole blood and dried biofluid swab samples, displaying its compatibility with sample types frequently found in forensic casework.
The outcomes from this study suggest the potential of HTG EdgeSeq microRNA Whole Transcriptome Assay to be employed in forensic biofluid identification, as it demonstrates high reproducibility, multiplex capacities, and compatibility with common forensic casework samples. Future studies encompassing a broader spectrum of biofluid types, and a greater number of individuals are required to validate the findings in this study and optimize the workflow for applying the assay in forensic laboratories.