Department of Physics, Chemistry and Biology (IFM)

The number of mtDNA copies per diploid nucleus in mouse tissues was determined using real-time PCR absolute quantification, using an ABI 7500 Fast system (Applied Biosystems). Total genomic DNA was purified from mouse tissues using the DNeasy Blood and tissue kit (QIAGEN). 5 to 10 ng of genomic DNA were used in each reaction. Primers and probe for mouse mt-ND1 gene (mitochondrially encoded NADH dehydrogenase 1) and for single-copy mouse RPPH1 gene (nuclear encoded ribonuclease P RNA component H1) were designed for this purpose (given below). For each DNA sample, the mitochondrial gene mt-ND1 and the nuclear gene RPPH1 were quantified separately. Standard curves have been done using known copies of a plasmid containing one copy of each of those two mouse genes referred above. According to the standard curve, the number of copies from each gene was calculated for each sample and the number of mtDNA copies per diploid nucleus was calculated according to the formula:

mtDNA copies per diploid nucleus = 2 x (mt-ND1 gene copies/RPPH1 gene copies)

PRIMERS

RPPH1

Fw : 5’-GGAGAGTAGTCTGAATTGGGTTATGAG

Rv : 5’-CAGCAGTGCGAGTTCAATGG

mt-ND1

Fw : 5’-TCGACCTGACAGAAGGAGAATCA

Rv: 5’-GGGCCGGCTGCGTATT
 

PROBES

RPPH1   :FAM-CCGGGAGGTGCCTC-TAMRA

mt-ND1           :FAM-AATTAGTATCAGGGTTTAACG-TAMRA