Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization

Abstract

A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus–infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin‐labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc.

Analysis of Mitochondrial DNA Polymorphisms in the Human Cell Lines HepaRG and SJCRH30

“…Importantly, HepaRG displays sensitivity to hepatotoxic compounds such as acetaminophen and aflatoxin B 1 [25,26]. Recent studies support that HepaRG is a suitable model to test drug-induced mitochondrial toxicity and mtDNA homeostasis [3,26,27,28]. Here we report the mtDNA genome polymorphisms and heteroplasmy of SJCRH30 and HepaRG.…”

“…More than 1000 human mitochondrial proteins are encoded by the nuclear genome and must be imported into mitochondria following translation on cytoplasmic ribosomes [1]. The mitochondrial DNA genome (mtDNA) is an ~16.6 kilobase pair (kbp) covalently closed circular molecule that contains 13 genes for polypeptides, 2 genes for rRNAs, and 22 genes for tRNAs [2,3,4]. Our maternally inherited mtDNA is critical to cellular viability as exemplified by the numerous disease mutations associated with it and by observations that knocking out mtDNA maintenance genes results in embryonic lethality in various mouse models [5,6,7].…”

The antiretroviral 2′,3′-dideoxycytidine causes mitochondrial dysfunction in proliferating and differentiated HepaRG human cell cultures

“…Differentiated HepaRG DNA samples were prepared from cells obtained 7 days post-differentiation. BamHI-digested human genomic DNA samples generate a 16.6-kb mtDNA genome-length band and a 2.2-kb nDNA band as described ( 79 ). Using the relative comparison of the MT to the N probe, differentiated HepaRG cells contain ∼2-fold more mtDNA than proliferating cells.…”

“…Except for blots containing samples obtained from proliferating HepaRG at day 13, mtDNA content was measured on blots that were simultaneously probed with the 18S nDNA probe (N) and the mtDNA-specific probe (MT) as described in the legend for Figure 1 , and the mean normalized band intensity values of the vehicle control samples (0 μM ddC) were set to 100%. Dual-probed blots containing day 13 samples from proliferating HepaRG treated with 1 μM could not be quantitated owing to low mtDNA signal; however, when the blots were stripped and probed individually with the mtDNA probe, stripped again, and then probed with the 18S probe, 0 and 1 μM mtDNA bands could be quantitated in Fiji as described ( 79 ). To confirm equal loading of whole-cell extracted DNA into each lane, the %CV for the nDNA bands on the single-probed 18S blots were at most 11% (100% ± 11% for 0 μM; 102% ± 10% for 1 μM; 101% ± 8% for 12 μM, errors are SDs), and the 0, 1, and 12 μM ddC–treated 18S sample band intensities are not significantly different from one another as judged by a one-way ANOVA.…”

“…Nuclear DNA (nDNA)- and mitochondrial DNA (mtDNA)-specific probes used for Southern blotting and immunodetection of target DNA fragments were synthesized via PCR using the PCR DIG Probe Synthesis Kit (Roche) as described ( 79 ). Briefly, each probe was synthesized separately from their respective plasmid templates.…”