Hi Timothy,

we isolated genomic and mitochondrial DNA from peripheral nerves and from various tissues of the CNS and quantified the ratio between mtDNA and gDNA by qPCR. The results were very robust when we compared the individual mice with the same genotype. This would be the protocol we used:

Mitochondrial DNA and genomic DNA from tissue were purified by using genomic DNA isolation Kit and G20 columns (Qiagen). DNA was resuspended in Tris-EDTA (TE) buffer, sonicated in a water-bath for 10 min and dissolved over night. The quantitative PCR was performed in two different dilutions of DNA input (between 5 ng and 160 ng).

The following review was really helpful: Malik AN, Czajka A. Is mitochondrial DNA content a potential biomarker of mitochondrial dysfunction? Mitochondrion. 2012 and our results were just published online in BRAIN.

Cheers,

Axel

Strictly speaking, mtDNA content is a poor marker for mitochondrial content. E.g. rho-0 cells have no mtDNA, and yet they do have mitochondria.

Dear Timothy, you can isolate total RNA and measure by real-time PCR with primers against specific mRNA that are generated in mtDNA. Then, you can detect mRNA expression of all 13 genes encoded in the mitochondria, comprising 7 subunits of complex I (NADH dehydrogenase; ND1 to ND6), 1 subunit of complex III (cytochrome c oxidoreductase; cyt b), 3 subunits of complex IV (cytochrome c oxidase; COX1 to COX3) and 2 subunits of complex V (ATP synthase; atp6 and atp8). I suggest to read the following paper: Koyanagi M et al. Plos One 2011; "Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1". Best regards and good luck!!!

As with Axel, we have also used qPCR for this, but from mammalian cell lines, with the results expressed as a ratio of mtDNA to nucDNA. We used mtDNA specific and nucDNA (single copy gene) specific primers in separate reactions and SYBR green. You can control the amount of total DNA you add to the qPCR, but since the ratio of mt to nuc is unknown, and the two loci are likely amplifying with different efficiency, you don't really know how much of either you have. But you can see how the ratio changes compared to a control (e.g., no treatment) or over time. One thing we thought about, but never did, was to make a sort of "standard curve", where you could input a known amount of pure mtDNA. This could be done by cloning the PCR product or a chunk of mtDNA that encompasses it. Assuming you can purify this plasmid well and quantify it, you could make serial dilutions with known copy numbers of the plasmid, calculated from the m.w. That would seem to allow you to express your qPCR results in terms of copy numbers of mtDNA.

Perhaps a functional assay, like oxygen consumption on homogenized tissue samples, would be appropriate, since it would be difficult to reconcile the mito’s significance in the tissues with EM, immunoblotting, or PCR.

I actually agree with Mikhail Alexeyev, mtDNA copy number is a poor marker of mt density... Alternatively, you can try to measure the citrate synthase activity which is a very simple spectrophotometric assay much more reliable... For further markers see http://onlinelibrary.wiley.com/doi/10.1113/jphysiol.2012.230185/full

Hi!

I'm afraid that what you're suggesting won't be accurate because a mitochondrion can have more than just one copy of its DNA (some times there are 10 or more copies) and this can vary from cell to cell and even inside the same cell. I suggest you to measure mitochondrial mass using a mitochondrial dye (such as Mitotracker) in a flow cytometer, it's better. good luck!!

We used the PCR-based method described by Kaaman et al. Diabetologia 50: 2526-2533, 2007.

I agree that qPCR can give a fairly accurate measure of the mtDNA/nuclear DNA ratio, but does not necessarily reflect the "number " of mitochondria. In view of the constant fusion and fission, a "number" may also not reflect mitochondrial activity. Moreover, a very important parameter is the cristae density, i.e the total area of the inner membrane, which may or may not be proportional to the activity of the OXPHOS machinery.

In our work (e.g., Hasek et al. Diabetes 62: 3362-3372, 2013) we were working with white adipose tissue and combined the PCR-based method referenced above with measures of citrate synthase activity, measures of cell area, and measures of fatty acid oxidation. In this setting, the doubling of the mtDNA/genomic DNA ratio was strongly correlated with increased oxidative capacity, reduced cell size, and remodeling of cell morphology. The strength of these relationships differs among tissue and cell types.

A detail to watch out for: Primers targeting mitochondrial DNA will nearly always also amplify nuclear DNA, because multiple copies of mtDA reside in the genome (in both mice and men). So whichever primer you want to use, published or self-designed, make sure the ones for mtDNA are really specific for that (e.g. with an electronic PCR or BLAST/BLAT of the predicted amplicon).

Try with this kit, http://www.abcam.com/mitochondrial-dna-isolation-kit-ab65321.html

Did you try to perform experiments with PGC1 alpha? Is the master regulator of mitochondrial biogenesis. You must have this analysis regardless of DNA mt quantification, which it is ok. Unfortunately I do not have any protocol for DNAmt.

I agree with Immo Scheffler. Using qPCR to measure the mtDNA/nDNA ratio gives you rather an indication of mitochondrial function. mtDNA content measurements can be strengthened in combination with expression analysis of master regulators of mitochondrial biogenesis as Agustina Alaimo suggested (see also Sahin et al. 2011 Nature). In our (epidemiological) studies, we use mtDNA content (qPCR) as a marker of mitochondrial damage and dysfunction. Thus, I think a lot depends on your question, but in your case I think other more “accurate” methods are warranted such as cardiolipin content and citrate synthase activity.

Citrate synthase assay is very reliable, if you make sure to fully disrupt the mitochondrial membranes, as this is a matrix enzyme. If needed I can provide a good protocol for it. Cardiolipin content also works very well. Give a look to: http://www.ncbi.nlm.nih.gov/pubmed/22586215

I would kindly ask you to Immo Scheffler how to measure cristae density, this is very interesting. Thanks for any suggestion

Hi, Axel Niemann. I saw the protocol you used. It's helpful. I still wonder that which mitochondrial gene did you chose for the quantification PCR. Lots of papers are about the human mitochondria, so, if you have done the work in mice, could you show me your selection of the mitochondria gene and the primer pairs? Thanks a lot!

Mitochondrial DNA copy number dose not represent the mitochondrial activity. In my study I found it totaly oposit. The copy number showed very high in one of my groups in my mice cohort interestingly citratesynthase activity (which is kind of representative for the active mitochodria) showed very different almost oposit. But about the further study for measuring OXPHOS mito profile can be measured.