Determining Mitochondrial Copy Number

• D-Loop Primers (for determining the number of mitochondrial DNA copies)
  • Forward: GGC CCA TTA AAC TTG GGG GT
  • Reverse: TTC TTC ACC GTA GGT GCG TC
• DNA Primers (for determining the number of nuclear DNA copies). Can use any genotyping primers with an amplicon <200bp. We use the Tsc locus. Do not use exon spanning qPCR primers.
  • Forward AAG AAG CCT CTT CTG CTA CC
  • Reverse CAG CTC CGA CCA TGA AGT G

1. Prepare DNA according to Extraction of DNA from TRIZOL preparations
2. Run qPCR as normal, but skip the cDNA synthesis step and use the DNA extract directly
3. Normalized each sample to D-Loop/Nuclear values by the ΔΔCt method. This should give the number of mitochondrial copies per nuclear DNA copy.
4. Divide this by two for diploid cells/tissues to get the number of mitochondria per cell

Stephenson EJ, Ragauskas A, Jaligama S, Redd JR, Parvathareddy J, Peloquin MJ, Saravia J, Han JC, Cormier SA, Bridges D. Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice. Am J Physiol Endocrinol Metab. 2016 Jun 1;310(11):E1003-15. doi: 10.1152/ajpendo.00521.2015. Epub 2016 Apr 26. PMID: 27117006; PMCID: PMC4935140.