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
Brian Davis
Committee Member
Glen Jackson
Abstract
Extraction methods which recover the maximal amount of DNA for downstream analysis are critical for casework samples. Additionally, with growing interest in using mRNA for body fluid identification and donor assignment via cSNPs, RNA must also be co-extracted with DNA. Furthermore, if DNA and RNA are highly compromised, proteins can potentially be used for body fluid identification and individualization using single amino acid polymorphisms (SAPs) resulting in genetically variant peptides (GVPs). A method capable of simultaneously extracting all these analytes from trace biological samples is needed. We have developed a method which uses protamine-conjugated paramagnetic beads to bind DNA and RNA, while keeping proteins intact within the supernatant. DNA recovery using 1 µL semen and 2 µL saliva was comparable to a commercial bead-based DNA extraction kit (~65 ng and 6 ng, respectively), but amounts recovered from 1 µL peripheral blood were lower (~5 ng instead of 19 ng). To enhance recovery of DNA from peripheral blood, we tested the incorporation of several additives during extraction including polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and dimethyl sulfoxide (DMSO). We also tried pH and temperature modulation in addition to bead titration. Although still lower than with the commercial kit, DNA recovery was significantly enhanced using an increased lysis temperature of 56 °C and an increased bead amount to 600 µg per extraction. RNA recovery was consistently higher with our method, and both DNA and RNA recovered were successfully used for DNA and mRNA profiling with the added advantage of retaining proteins suitable for identification using LC-MS/MS.
A full developmental validation, in accordance with Scientific Working Group of DNA Analysis Methods (SWGDAM) guidelines was conducted on this novel method, and results from repeatability, reproducibility, and stability parameters are presented. Repeatability and reproducibility tests included two users extracting DNA, RNA, and protein from 1 µL peripheral and menstrual blood, 1 µL semen, 20 µL saliva, and 5 µL vaginal secretion (from 300 µL PBS containing a vaginal swab) on two separate days. All three analytes were quantified and the coefficient of variation percentage (% CV) was calculated. DNA % CV estimates across both users and both days were ≤31% for all body fluids except saliva (42%) and semen (54%). For RNA, % CV estimates were higher, with peripheral blood, vaginal secretion, and saliva estimates being ≤36% while menstrual blood and semen estimates were 56% and 58%, respectively. The % CV estimates for proteins were all ≤27%, except for semen at 49%. Additionally, full GlobalFiler™ profiles were obtained, body fluid specific mRNA markers were amplified, and several body fluid specific proteins were identified in all samples.
For the stability tests, we used samples containing inhibitors and degraded DNA in extractions. For inhibitor studies, 1 µL semen on cotton was mixed with either 0.5 µL peripheral blood to model hematin or soil to model humic acid. Samples were either extracted immediately or stored for 1 week before extraction. Quantifiler® Trio IPC Ct values indicated removal of inhibitors from extracts, but DNA and RNA recovery were much lower for samples containing humic acid from soil. Full DNA profiles were generated from all samples containing hematin from blood, but with samples containing humic acid from soil, full profiles were only obtained in 4/6 samples. The semen specific mRNA marker (SEMG1) was detected in 4/6 and 2/6 hematin and humic acid replicates respectively, but 11 to 13 semen specific proteins were identified in all supernatants using LC-MS/MS. For degraded DNA studies, moderately (~500 bp) or highly degraded (~150 bp) DNA was deposited onto various substrates (cotton fabric, glass slide, polypropylene tube, or brass doorknob) and stored for either 1 week or 1 month. Recovery of DNA was highest from cotton fabric, at 89% and 97% using the 500 bp DNA, and 61% and 52% using the 150 bp DNA after 1 week and 1 month, respectively.
Overall, results indicate that our method is suitable for extraction of DNA (even when severely degraded) and RNA from trace volumes of body fluids while also retaining proteins. Validation experiments have shown that the method is repeatable and reproducible among different users and times and can successfully remove inhibitors from samples. Once validated, this method, which has a simple workflow and is amenable to automation, will allow the entire suite of analytes to be analyzed for casework.
Recommended Citation
Hetzke, Jacob Alexander, "Optimization and developmental validation of a novel protocol for recovery of multiple analytes from trace biological samples - reproducibility, reliability, and stability studies" (2026). Graduate Theses, Dissertations, and Problem Reports. 13260.
https://researchrepository.wvu.edu/etd/13260