Towards molecular autopsies: Development of a FFPE tissue DNA extraction workflow. Issue 2 (March 2022)

• Record Type: Journal Article
• Authors: Viljoen, Rabia; Reid, Kate Megan; Mole, Calvin Gerald; Rangwaga, Mmannini; Heathfield, Laura Jane
• Is Part Of: Science & justice. Volume 62:Issue 2(2022)
• Page Start: 137
• Page End: 144
• Publication Date: 2022-03
• Subjects: Formalin fixed paraffin embedded tissue -- Sudden unexpected death -- Molecular autopsy -- DNA extraction optimisation -- Microscopic nuclei quantification; Forensic sciences -- Periodicals; Criminal investigation -- Periodicals; Forensic Medicine -- Periodicals; Jurisprudence -- Periodicals; Criminalistique -- Périodiques; Enquêtes criminelles -- Périodiques; Criminal investigation; Forensic sciences; Electronic journals; Periodicals; 363.2505
• DOI: 10.1016/j.scijus.2021.12.005
• Languages: English
• ISSNs: 1355-0306

Abstract

Highlights: The development of a workflow to extract DNA from FFPE tissue is described. The microscopic quantification of nuclei guided selection of tissue type to use. Using 100 tissue sections, each 1 µm in thickness, significantly improved DNA recovery. The QIAamp® DNA FFPE Tissue Kit outperformed the Zymo and Macherery-Nagel kits. Longer storage period (up to 3.5 years) did not significantly affect DNA integrity. Abstract: Sudden unexpected death (SUD) is a devastating event and forms a substantial proportion of the cases investigated at forensic mortuaries each year. Despite post-mortem investigations, the cause of death may remain undetermined. There is potential for these unresolved cases to benefit from retrospective molecular autopsies for investigation into genetic mutations which may have contributed towards death. Often, formalin fixed paraffin embedded tissues (FFPET) are the only archival sources of DNA available for retrospective analyses. However, extracting usable DNA from FFPET is challenging as current methods yield poor quality and quantity DNA. Thus, this study aimed to optimise DNA recovery from FFPET by investigating several variables within the DNA extraction workflow, including the selection of tissue type, number and thickness of tissue sections, deparaffinisation method, and DNA extraction kit. The quantity and quality of DNA recovered were assessed using spectrophotometry, real time PCR, digital capillary electrophoresis and DNA profiling. This study was the first to implement a nuclei quantification using microscopy to guide the selection of the best tissue type to use for DNA analysis. The use of a greater number of thinner tissue sections (100 sections, each 1 μm) significantly improved DNA concentration, purity and fragment length. Additionally, the combination of Deparaffinization Solution with the QIAamp® DNA FFPE Tissue Kit proved most favourable with a median DNA yield of 320 ng and 55% of DNA fragments greater than 400 bp. Isolated DNA was of single source, indicating no contamination in the workflow, and FFPET blocks that were stored for up to 3.5 years did not significantly affect DNA degradation ( p = 0.1764). These results are especially informative for designing library preparation and sequencing workflows for determining cause of death in unresolved SUD cases.