Biological and translational attributes of mitochondrial DNA copy number: Laboratory perspective to clinical relevance

Table 2 Methods for assessing mitochondrial DNA copy number

Method	Steps involved	Applications	Advantages	Limitations	Ref.
qPCR	DNA extraction → primer design → amplification → analysis	Widely used in clinical and research diagnostics	High sensitivity, high throughput, cost-effective, rapid	Susceptible to bias in low-quality DNA, issues with heterogeneous samples, requires careful primer design	[15,24-26]
NGS	DNA extraction → library preparation → sequencing → bioinformatics analysis	Genome-wide studies, detects mtDNA mutations alongside copy number analysis	Genome-wide analysis, accurate quantification, detects mtDNA heteroplasmy	Requires advanced bioinformatics, high cost, computational complexity	[21,28-32]
Southern blot hybridization	DNA extraction → gel electrophoresis → hybridization → quantification	Historically the gold standard, reliable for mtDNA integrity assessment	High reliability, detects large-scale deletions	Time-intensive, requires large DNA quantities, semi-quantitative	[22]
FISH	Sample preparation → probe hybridization → microscopy	Single-cell resolution studies, spatial visualization of mtDNA	Single-cell resolution, visualizes mtDNA distribution	Labor-intensive, provides only rough mtDNA estimates, technically demanding	[23]

qPCR: Quantitative polymerase chain reaction; NGS: Next-generation sequencing; FISH: Fluorescent in situ hybridization; mtDNA: Mitochondrial DNA.