Comparison of sorption methods for total DNA isolation from muscle tissue: influence on the yield and integrity of mitochondrial DNA
DOI:
https://doi.org/10.17308/sorpchrom.2025.25/13447Keywords:
DNA extraction, sorbents, silicon dioxide, magnetic particles, mitochondrial DNA, fragmentation, muscle tissueAbstract
Neuromuscular diseases associated with genetic and mitochondrial impairments require high-quality DNA extraction from muscle tissue for subsequent molecular genetic analysis. This study presents a comparative evaluation of total DNA extraction methods from mouse skeletal muscle, including precipitation, silica membrane adsorption, bulk sorbents, and magnetic particles. The results demonstrated that although precipitation yielded the highest total DNA amount, the proportion of mitochondrial DNA (mtDNA) in these samples was minimal due to non-selective co-precipitation of degraded nuclear DNA fragments. In contrast, column-based adsorption and magnetic particle methods provided significantly higher mtDNA content (4.5-fold increase) and better preservation of mitochondrial genome integrity. Bulk sorbents showed the highest level of mtDNA damage, likely due to mechanical fragmentation and suboptimal elution conditions. Quantitative real-time PCR with SYBR Green proved to be the most effective method for assessing mtDNA integrity, enabling precise detection of even minor lesions, unlike qualitative approaches. Notably, SYBR Green did not inhibit amplification of ~2 kbp fragments, confirming its suitability for mtDNA analysis. The highest vulnerability to damage was observed in the mtDNA D-loop region, a key regulatory site, which is particularly relevant for studying mitochondrial dysfunction in muscle tissue. Thus, column-based or magnetic particle sorption methods combined with quantitative PCR are optimal for mtDNA studies, ensuring high sensitivity and reliability of results.
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