Directed Research Project
Tracey Dawson Green
Traditional enzymatic tests for body fluid identification are prone to false negatives, false positives, and several body fluids do not have confirmatory or reliable presumptive tests. Therefore, molecular-based tests may be more reliable, such as messenger RNA (mRNA), microbial DNA, or microRNA (miRNA) assays.
miRNAs are small, noncoding RNAs whose main function in-vivo is the regulation of protein expression by selectively suppressing the translation of their corresponding mRNAs. They also lack a poly-A tail, and because of this, their small size, and their association with other molecules such as the RISC protein complex, they are more robust than other RNAs, and more easily detected in compromised samples. miRNAs are also present in DNA extracts.
The overall purpose of this project is to test the robustness of an optimized miRNA panel as part of the developmental validation process. This panel was tested within individuals over a biological cycle, and samples that have been exposed to either a heat, chemical, UV, or environmental treatment. Detection of these markers was evaluated using reverse-transcription quantitative PCR (RT-qPCR). Following RT-qPCR, the differential expression values (ΔCq) were calculated and input into a quadratic discriminate analysis model for body fluid classification.
Significant differences were present in the detection of these markers within the same donor in saliva and blood, however, all body fluids except for blood and urine exhibited low classification rates compared to previous population studies, especially menstrual blood, which was often incorrectly classified as vaginal fluid. Blood and urine that were treated with heat both showed classification rates similar to previous population studies. In contrast, semen and saliva both showed a low correct classification rate. The classification rate of each body fluid after either a chemical or UV treatment depended on both the body fluid and the marker. Dish soap, 1:10 bleach and full-strength bleach treatment impacted the detection of miRNA markers in semen. Bleach affected the detection in saliva, glacial acetic acid affected detection in urine, and all treatments except UV affected the detection of markers in blood, although not enough to affect the classification rate. The environmentally treated samples were all correctly classified in blood and urine, however, neither semen nor saliva were correctly classified in any of the samples due to failure to amplify. Overall, the classification rates in compromised samples were similar to population studies in blood and urine and were lower in semen and urine.
In future studies, this assay could benefit from adding markers of different types, such as microbial DNA markers, and using high throughput sequencing to increase its multiplexing ability, which would decrease the required analyst time. Although vaginal fluid, saliva, and semen showed relatively low classification rates, the other body fluids tested were resistant to degradation and showed stable detection within a donor. This assay could prove to be a reliable, time efficient method for identifying any forensically relevant body fluid.
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
Available for download on Saturday, May 06, 2023