Formalin-fixed, paraffin embedded (FFPE) tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research and clinical applications. However, FFPE DNA extraction presents unique challenges and requires lengthy incubation periods, which can be impractical for certain applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE generates a dense bubble cloud of acoustic cavitation capable of ablating tissue into an acellular lysate. FUSE treatment was applied to de-paraffinized porcine pancreas FFPE scrolls, followed by heated incubation for formaldehyde-induced DNA-protein crosslink reversal. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity ratios compared to conventional methods. DNA extracted via FUSE showed comparable fragmentation to conventional methods, and three out of four samples successfully amplified via PCR, indicating suitability for downstream analysis. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications. / Master of Science / Formalin-fixed, paraffin embedding (FFPE) has historically been the most popular method of biological tissue preservation, as it allows tissue to remain shelf stable for decades. As such, FFPE tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE treatment applies rapid, focused ultrasound waves to tissue, resulting in the mechanical breakdown of cells and subsequent release of DNA. FUSE treatment was applied to pig pancreatic FFPE samples. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity compared to conventional methods. Three out of four DNA samples extracted via FUSE were successfully amplified, and DNA fragment lengths were comparable between FUSE and conventional methods, showing that FUSE did not fragment DNA beyond useful fragment lengths. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/115718 |
| Date | 10 July 2023 |
| Creators | Mehochko, Isabelle Grace |
| Contributors | Department of Biomedical Engineering and Mechanics, Vlaisavljevich, Eli, Holmes, Hal, Allen, Irving Coy |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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