title: Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Life Science and Medicine, King's College London, SE1 1UL, UK. Electronic address: afshan.malik@kcl.ac.uk.
authors.forename: Afshan N
authors.initials: AN
authors.lastname: Malik
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Life Science and Medicine, King's College London, SE1 1UL, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Czajka
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Life Science and Medicine, King's College London, SE1 1UL, UK.
authors.forename: Phil
authors.initials: P
authors.lastname: Cunningham
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics; D008297:Male; D008810:Mice, Inbred C57BL; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial
keywords: Mitochondrial DNA; Mitochondrial pseudogenes; Mouse mitochondrial genome; NumtS; Real time PCR