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Updated: Mar 21, 2026
Methodology for Accurate Detection of Mitochondrial DNA Methylation
Published on: May 20, 2018
Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion
Afshan N Malik1, Anna Czajka1, Phil Cunningham1
1Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Life Science and Medicine, King's College London, SE1 1UL, UK.
Background:
Mitochondria contain an extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes in MtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification of MtDNA and to establish a method designed to avoid this.
Methods:
The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NumtS were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclear genome using real time qPCR and absolute quantification.
Results:
Approximately 95% of mouse MtDNA was duplicated in the nuclear genome as NumtS which were located in 15 out of 21 chromosomes. A unique region was identified and primers flanking this region were used. MtDNA levels differed significantly in mouse tissues being the highest in the heart, with levels in descending order (highest to lowest) in kidney, liver, blood, brain, islets and lung.
Conclusion:
The presence of NumtS in the nuclear genome of mouse could lead to erroneous data when studying MtDNA content or mutation. The unique primers described here will allow accurate quantification of MtDNA content in mouse models without co-amplification of NumtS.
Insights
Mouse nuclear mitochondrial insertion sequences (NumtS) can interfere with mitochondrial DNA (MtDNA) quantification. Researchers developed new primers to accurately measure MtDNA levels in mouse tissues, overcoming NumtS interference for reliable biomarker studies.
Area of Science:
- Genomics
- Molecular Biology
- Biochemistry
Background:
- Mitochondrial DNA (MtDNA) plays a crucial role in cellular energy production.
- MtDNA damage is linked to inflammation and bioenergetic deficits, making MtDNA levels a key biomarker for mitochondrial dysfunction.
- Nuclear mitochondrial insertion sequences (NumtS), which are fragments of MtDNA integrated into the nuclear genome, can interfere with accurate MtDNA quantification, as previously observed in humans.
Purpose of the Study:
- To investigate the impact of mouse NumtS on mitochondrial DNA (MtDNA) quantification.
- To develop and validate a method for accurate MtDNA copy number determination in mouse tissues, free from NumtS interference.
Main Methods:
- Confirmation of mouse NumtS using BLAST N and identification of unique MtDNA regions via FASTA.
- Design and testing of specific MtDNA primers to avoid co-amplification of NumtS.
- Quantification of MtDNA copy numbers in various mouse tissues using real-time qPCR and absolute quantification, expressed as a ratio of mitochondrial to nuclear genomes.
Main Results:
- Approximately 95% of mouse MtDNA is duplicated in the nuclear genome as NumtS, distributed across 15 of 21 chromosomes.
- A unique MtDNA region was identified, and primers targeting this region were successfully developed.
- Significant variations in MtDNA levels were observed across mouse tissues, with the highest concentrations in the heart, followed by kidney, liver, blood, brain, islets, and lung.
Conclusions:
- The presence of NumtS in the mouse nuclear genome can lead to inaccurate results in studies of MtDNA content and mutations.
- The novel primers presented in this study enable precise quantification of mouse MtDNA content, effectively preventing NumtS co-amplification and ensuring reliable data for mitochondrial research.
