Table 2.

A summary of the major issues and potential solutions in the field of mitochondrial epigenetics.

Caveat	Potential issues	Potential solutions	Ref.
Genetic issues	Incorrect determination of pseudogenes as mtDNA affects the validity of results Genetic mutations in mtDNA may have specific associated methylation signatures	Isolate mitochondria before mtDNA extraction to avoid nuclear contamination Specific primers designed with the consideration of NUMT amplification BLAST search to identify known NUMTs Haplogroup and heteroplasmy studies should consider mtDNA methylation as a potential variable	[62]
Cell specificity and technical issues	Different brain regions have differential methylation patterns and different cell population compositions Reduced methylation levels in mitochondria and variation in tDNA copy number may increase noise and dilute signals Bisulfite-based methodologies cannot distinguish between 5-mC and 5-hmC	Larger samples sizes in specific brain subregions will improve statistical significance FACS or LCM to separate cell types such as glia and neurons prior to analysis Comparative analysis of techniques for their suitability to mitochondrial methylation studies should be considered Using oxidative bisulfite-sequencing allows for the distinction of 5-mC and 5-hmC at single base resolution	[63]

5-hmC: 5-hydroxymethylcytosine; 5-mC: 5-methylcytosine; FACS: Fluorescence-activated cell sorting; LCM: Laser capture microdissection; NUMT: Nuclear mitochondrial pseudogene.