Mitochondrial DNA depletion analysis by pseudogene ratioing

Abstract

The mitochondrial DNA (mtDNA) depletion status of ρ 0 cell lines is typically assessed by hybridization or polymerase chain reaction (PCR) experiments, in which the failure to hybridize mtDNA or amplify mtDNA using mtDNA-directed primers suggests thorough mitochondrial genome removal. Here, we report the use of an mtDNA pseudogene ratioing technique for the additional confirmation of ρ 0 status. Total genomic DNA from a U251 human glioma cell line treated with ethidium bromide was amplified using primers designed to anneal either mtDNA or a previously described nuclear DNA-embedded mtDNA pseudogene (mtDNAψ). The resultant PCR product was used to generate plasmid clones. Sixty-two plasmid clones were genotyped, and all arose from mtDNAψ template. These data allowed us to determine with 95% confidence that the resultant mtDNA-depleted cell line contains less than one copy of mtDNA per 10 cells. Unlike previous hybridization or PCR-based analyses of mtDNA depletion, this mtDNAψ ratioing technique does not rely on interpretation of a negative result, and may prove useful as an adjunct for the determination of ρ 0 status or mtDNA copy number.