Author ORCID Identifier
Semester
Spring
Date of Graduation
2026
Document Type
Thesis
Degree Type
MS
College
Eberly College of Arts and Sciences
Department
Forensic and Investigative Science
Committee Chair
Arati Iyengar
Committee Member
Glen Jackson
Committee Member
Brian Davis
Abstract
Conventional forensic biology methods prioritize the extraction of DNA from biological samples while other macromolecules are neglected or discarded in the process. In many cases, identification of the body fluid present and linking the DNA profile to the body fluid is essential to provide context. This is increasingly being achieved using body fluid specific mRNA markers, making co-extraction of RNA with DNA from trace biological samples highly desirable. Further, DNA may be compromised or completely absent from a sample, whereas more robust proteins may still be present. Proteins are also useful for body fluid identification and offer potential for human identification through genetically variant peptides (GVPs) but are destroyed and discarded in conventional DNA extraction protocols.
We have developed a novel extraction method that simultaneously isolates DNA, RNA, and proteins from trace biological samples using protamine conjugated paramagnetic beads to bind DNA and RNA, while retaining proteins unbound in the supernatant. Trace amounts of five body fluids commonly encountered in forensic science, including peripheral blood, menstrual blood, semen, saliva, and vaginal secretion were successfully used to extract DNA, RNA, and proteins. DNA was quantitated using the Quantifiler™ Trio DNA Quantification Kit, and the GlobalFiler™ PCR Amplification Kit was used for STR DNA profiling. RNA was quantitated using the QuantiFluor® RNA System, and body fluid specific mRNA amplification was performed, followed by capillary electrophoresis. Proteins were quantitated using the Pierce™ Detergent Compatible Bradford Protein Assay Kit, with selected samples outsourced for protein identification.
A full developmental validation was performed on this method according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. Specific parameters tested in this study include sensitivity, mixtures, and case type samples. Sensitivity was tested separately for DNA, RNA, and proteins across all five body fluids, and results show that our multi-analyte extraction method is capable of extracting DNA, RNA, and proteins from a wide range of input volumes, from as low as 0.008 μL of peripheral blood, 0.016 μL of menstrual blood, 0.01 μL of semen, 0.02 μL of saliva, and 0.05 μL of vaginal secretion (from a swab placed in 300 μL of PBS). A range of case type samples were prepared for each body fluid using different substrates (cotton fabric, plastic tubes, glass slides, and metal doorknobs) at two time points, 1 week and 1 month. Results demonstrate successful extraction of DNA, RNA, and proteins from 1 µL of peripheral blood, 1 µL of menstrual blood, 1 µL of semen, 20 µL of saliva, and 5 µL of vaginal secretion (from a swab placed in 300 µL of PBS) applied to various substrates after both time points. Downstream analysis demonstrated successful STR DNA profiling, detection of mRNA markers ALAS2, SEMG1, and HTN3, and identification of several body fluid specific proteins. Seven mixtures of body fluids at two different ratios (1:5 and 1:10) were tested for recovery of multiple analytes from the minor male and major female contributors. DNA from the minor male contributor was successfully extracted from all seven mixtures. In most cases, the mRNA markers from both the male and female contributors were detected, and 4 – 45 body fluid specific proteins were identified for each contributor in all mixture types.
Finally, we determined that our extraction method is non-destructive to drugs and metabolites by spiking cocaine and its metabolites, benzoylecgonine and methylecgonine, into saliva extractions and using solid phase extraction (SPE) followed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to detect the drug and metabolites post extraction. Recovery of cocaine, benzoylecgonine, and methylecgonine was 56 – 71%, 60 – 82%, and 14 – 44%, respectively. Full DNA profiles were also obtained, and a saliva specific mRNA marker (HTN3) was successfully identified. This method will be the first multi-analyte extraction method to allow retention of the entire suite of analytes from trace biological samples, offering forensic scientists additional opportunities to analyze evidence.
Recommended Citation
Waggoner, Alexis Ann, "Developmental Validation of a Novel Multi-Analyte Extraction Method for Trace Biological Samples – Studies on Sensitivity, Mixtures, and Case Type Samples" (2026). Graduate Theses, Dissertations, and Problem Reports. 13258.
https://researchrepository.wvu.edu/etd/13258