microRNAs (miRNAs) are small noncoding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRNAs, when fully evaluated, will make excellent candidates for body fluid identification. miRNA analysis of body fluids has been the subject of some recent interest in the forensic community. In this study, small RNAs were isolated from individual donations of eight forensically relevant biological fluids (blood, semen, vaginal fluid, menstrual blood, saliva, urine, feces, and perspiration) and subjected to next generation sequencing using the Illumina Hi-Seq platform. Sequencing reads were aligned and annotated against miRbase release 21, resulting in a list of miRNAs and their relative expression levels for each sample analyzed. Body fluids with high bacterial loads (vaginal fluid, saliva, and feces) yielded relatively low annotated miRNA counts, likely due to oversaturation of small RNAs from the endogenous bacteria. Both body fluid specific (miRs-200b, 1246, 320c, 10b-5p, 26b, and 891a) and potential normalization miRNAs (let-7g and i) were identified for further analysis as potential body fluid identification tools for each body fluid.

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.

Molecular methods for body fluid identification have been extensively researched in the forensic community over the last decade, mostly focusing on RNA-based methods. Microbial DNA analysis has long been used for forensic applications, such as postmortem interval estimations, but only recently has it been applied to body fluid identification. High-throughput sequencing of the 16S ribosomal RNA gene by previous research groups revealed that microbial signatures and abundances vary across human body fluids at the genus and/or species taxonomic level. Since quantitative PCR is still the current technique used in forensic DNA analysis, the purpose of this study was to design a qPCR multiplex targeting the 16S gene of Bacteroides uniformis, Streptococcus salivarius, and Lactobacillus crispatus that can distinguish between feces, saliva, and vaginal/menstrual secretions, respectively. Primers and probes were designed at the species level because these bacteria are highly abundant within their respective fluid. The validated 16S triplex was evaluated in DNA extracts from thirty donors of each body fluid. A classification regression tree model resulted in 96.5% classification accuracy of the population data, which demonstrates the ability of this 16S triplex to presumptively identify these fluids with high confidence at the quantification step of the forensic workflow using minimal input volume of DNA extracted from evidentiary samples.

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