The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

• Published in: Mitochondrion 2021-11, Vol.61, p.179-187
• Main Authors: Repolês, Bruno Marçal, Gorospe, Choco Michael, Tran, Phong, Nilsson, Anna Karin, Wanrooij, Paulina H.
• Format: Article
• Language: English
• Subjects: DNA integrity; Long-range PCR; Mitochondrial DNA; mtDNA; Nuclease activity
• ISSN: 1567-7249; 1872-8278; 1872-8278
• DOI: 10.1016/j.mito.2021.10.005

•The procedure chosen for isolation of mtDNA from solid tissues has a considerable effect on the integrity of the product, as routine isolation methods yield mtDNA with numerous single-stranded nicks per mtDNA strand.•Modifications that benefit mtDNA integrity are made to the standard total DNA isolation procedure.•Four different DNA isolation methods are systematically compared with regard to the integrity of the resulting mtDNA, yield, mtDNA enrichment and labor.•We show that the choice of isolation method affects the performance of the mtDNA in downstream analyses such as long-range quantitative PCR. The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.