DOI: 10.3886/icpsr38391.v1
title: Verification and Evaluation of a miRNA Panel for Body Fluid Identification Using DNA Extracts, United States, 2019-2021
title: Version 1

description: Although human identification through DNA analysis has reached a level of maturity in the Forensic Science field with regards to the sophistication of the techniques and confidence in the results, the equally important question of body fluid identification has lagged behind, and could still be considered to be in a rudimentary state. Current crime scene and in-laboratory methods utilize detection methods that exploit the properties of each biological fluid (e.g. phenolphthalin or TMB testing for blood, amylase detection for saliva, and urease tests for urine), but validated confirmatory techniques are largely limited to microscopic methods (i.e. identification of spermatozoa) or immunological methods, as seen in the widely used immunochromatographic commercial tests for blood, semen, and other biological fluids.
Thus, while there is widespread confidence in the DNA profile generated, there is often significantly less assurance in the identity of the body fluid that the DNA profile was developed from. It is common during trials for attorneys to categorically accept the STR analysis, but probe the forensic scientist on the source of the DNA that generated the profile. Because of this dichotomy, significant efforts have been made over the past fifteen years in order to develop forensic serological techniques of a more discriminatory nature.
Of late, there has been some work in the forensic science field in regards to exploring microRNAs (miRNAs) for a molecular-based, forensic body fluid identification method. MiRNAs are small structures that are 19-23 nucleotides long and regulate cellular processes through interactions with mRNA by regulating gene expression through translational suppression or cleavage of a targeted mRNA. miRNAs are highly conserved among organisms, indicating their importance in regulating biological processes. As such, some miRNAs can be consistently expressed in all human tissues, and others can be tissue-specific Because of the potential for tissue specificity, their small size and consequent inherent stability, miRNAs have been the subject of recent research interest as a potential forensic body fluid identification technique. They are found in extracellular fluids, and thus the application of unique miRNAs for forensically relevant body fluids is a distinct possibility.
description: The purpose of this study was to build on previous work in the laboratory, wherein researchers identified a panel of 8 miRNAs that can identify blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions.
description: <b>Specific Aim 1: Evaluate existing miRNA markers in DNA extracts</b>

In this aim, researchers analyzed miRNA detection in DNA extracts via RT-qPCR analysis using a large population sample set in order to evaluate miRNA expression between body fluids in DNA extracts. Each candidate miRNA was tested with 50 population samples of each biological fluid. The data was used to redefine and retest the decision tree for body fluid identification previously optimized for RNA extracts, and additional markers were tested to improve prediction accuracy.


<b>Specific Aim 2: Evaluate miRNA panel performance in DNA extracts</b>

In this aim, researchers took the miRNA panel and analysis method optimized in Aim 1, and tested sets of samples from the same donor over biological time, as well as limit of detection studies, human organ/tissue cross-reactivity and species specificity studies. Stability studies using chemically and environmentally compromised samples addressed the potential for impact of differential degradation of the target miRNAs.
description: Response Rates: 93.1% in body fluid classification
description: Presence of Common Scales: None
description: Researchers evaluated a sample set of DNA extracts (50 donors for each body fluid) and evaluated miRNA expression using the method previously validated for RNA extracts using RT-qPCR analysis. Blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions were collected in accordance with the approved Institutional Review Board Human Subjects Research Protocol. Menstrual, fecal, and vaginal samples were collected on sterile cotton swabs by the donors and returned in swab boxes. Blood was deposited onto a sterile cotton swab after sterilizing and pricking the donor's finger with a Unistik 3 Normal lancet, and saliva was collected by rolling a sterile cotton swab along the inside of the donor's cheek. Urine and semen were deposited into sterile collection cups supplied to the donor, which were returned on ice within 24 hours before aliquoting each onto sterile cotton swabs (50 uL of semen and 100 uL of urine). All swabs were dried and stored in swab boxes at room temperature until DNA extraction.
description: Datasets: 
DS1: Verification and Evaluation of a miRNA Panel for Body Fluid Identification Using DNA Extracts, United States, 2019-2021
description: 50 unique donors of blood, semen, vaginal secretions, menstrual secretions, saliva, feces, and urine for miRNAs. Smallest Geographic Unit: Country
description: mixed mode