follow protocol given in kit by the manufacturer.

you can add DNase which would digest nuclear DNA followed by ultracentrifuge for isolating in pure...

Karuna Sagar@ is there any specific Dnase which only cleaves nuclear DNA?? I dont think addition of Dnase is not an good idea it can also cleaves mitochondrial DNA..

Karuna, I followed exactly the manufacturer's protocol, however it was not efficient for the effective isolation. I've thought about doing for an initial isolation of mitochondria, by ultracentrifugation, and then a conventional total extraction. What do you think?

If you need to isolate the whole mitochondrial genome, possibly for sequencing, then it might be a good idea to isolate the mitochondria first. There are quite a large number of papers out there describing methods for invertebrates. You might start here with this apper by Ead and Hands: Am J Physiol Regul Integr Comp Physiol 277:R1588-R1597, 1999

Oliver, thank you for you answer. In fact the methods described by Eads & Hand is almost the same of the ABICAM kit. I believe that this method leaves a considerable amount of nuclear DNA in the sample. Later in the quantification and also in sequencing (454) I could see that.

You could try a PCR with mitochondrial primers to preferentially amplify your mitochondrial DNA before 454 library prep. If you add 454 sequencing adaptors to the primer sequences then you can skip ligation and proceed directly to pooling and em-PCR.

Try going for organellar extractions and then DNA extraction from that mayb. Thats possibly the best fit for it.its a lengthy process with density gradient cell disruption and stuff ..but works out d best.kits r of no use till u do that.

I would isolate mitochondria then isolate mtDNA from them. Various kits available for mitochondrial tissue isolation or with the correct buffers you can do yourself, again many protocols available

I agree that you should isolate mitochondria, treat them with DNase, purify on sucrose cusion and then isolate mtDNA. Conditions of treatment and purification should be optimised for your invertebrates.

using sucrose purification on an extracted mitochondria treated with DNase and maintaining absoulte kit extraction protocols, a high through-put can be achieved.

Hello, I think Qiagen kits followed by column purification is the way to go, but you'll almost always get some nDNA in your samples. If you can design primer, I rather use Long-Range PCR (Promega polymerase is quite good).

Some isolation ptcols:

http://www.abcam.com/Mitochondrial-DNA-Isolation-Kit-ab65321.html

http://www.qiagen.com/products/protein/proteomics/qproteome/qproteomemitochondriaisolationkit.aspx

http://www.promokine.info/fileadmin/PDFs/All_PDFs/PK-CA577-K280.pdf

Kkkkit

We analyze freshly isolated mitochondrial from brain using the Seahorse XF-24

Thanks Hernan, it could be an option. I´ll try to buy this Qiagen kit.

use column based qiagen kit

What is the size of mitochondrial genome in this organism? Is it circular?

If it is circular, you can use exonuclease to digest (eliminate) linear DNA (nuclear DNA) and enrich for circular DNA.

QIAGEN column may specifically be designed for human Mitochondrial DNA (16-kb circular DNA) and thus may not be feasible for your organism.

That is the problem, we don´t know. It's a little-known group, but the mitochondrial genome may be circular and the size is certainly smaller than 19KB.

I thing gradient centrifuge is the way to go forward to first isolate the mitochondria, then you could even use NAse to kill the nuclear DNA contamination and then from the isolated mitochondria toy could use a kit to isolate the DNA

Hi,

What do you need mtDNA for? If you´re interested in replication or structure NEVER use a kit NOR heat the sample to 55´C. For most other applications (including deep sequencing) enriched mtDNA from crude mitochondria is good enough, would not make it too complicated. Quality depends also on your source material - how much, how difficult to homogenize and mtDNA copy number. Generally large amounts of easily homogenizable high copy number tissue give high yields of good quality mtDNA.

For pure mtDNA, the classical method has been to use CsCl gradient centrifugation, but there are nicer and cleaner ways to get good quality mtDNA out, which is suitable for even EM. Some method comparisons and refs here:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857715/

Oh, and the kits for isolating mitochondria are awful. Save your money and use differential centrifugation with sucrose gradient step.

Note that there are several examples of "frutti di mare" which have polydispersed mt genomes, which may or might not be circular. So be open for all options! I would first run the sucrose-gradient isolated mtDNA (+/- restriction enzyme digestion of your choice) on 0.4% agarose gel and see how it behaves.

Cheers,

Jaakko

Hi,

Jaakko is perfectly right. One minor addition, Percoll gradients will also be equally swell applicable. In addition, if you wish to get rid of any non-mtDNA contamination you can treat the crude mitochondrial fraction with a nuclease (e.g. Benzonase) for 15 min on ice followed by ProtK for 30 min on ice, 2 washes with PBS followed by gradient centrifugation. Of course, these additional incubation steps my impose a risk of losing some DNA-intermediates through degradation. It will be a cost and efficiency determination depending on what you are planning to do with the mtDNA.

Good luck,

Joachim

hi, for isolation of Mt DNA first you should go for density gradient centrifugation by using cesium chloride, then separate the Mt DNA upon density, then treat the supernatant containing Mt DNA with DNAse ( digestion of nuclear DNA) then follow as per the KIT or manual protocol.....

you should note that there are organisms, covering several non-Saccharomyces species from the yeasts for which you don´t get effiecient separation of nuclear and mtDNA. I would therefore refrain from using it on a standard basis - especially as in this case where the topology of the mtDNA etc. is still to be determined. Concerning kits, pls refer to Jaakko Pohjoismäki - safe your money.

Jaakko, thank you for your consideration. Indeed, I need to get a considerable amount (500ng) of mtDNA for sequencing on a 454. Now I have two options, the gradient centrifugation or full amplification of mtDNA using a LongPCR polymerase. I think I'll try both methods and then compare.

Dear Sergio, after almost 19 years working on mtDNA, I may state that the only efficient method is the separation separation under CsCl gradient. Please note that this is a very toxic reagent and requires ultracentrifugation. I have also experience with long PCR, but it is a very difficult approach concerning reproducibility... But when it works, is great! All the best!

Use the soft tissue of the animal and try standard homogenisation/differential contrifugation. Use a Dounce to break cells but not nuclei, get rid of nuclei with several rounds of low-speed centrifugation then extract DNA normally from supernatant and see what you get.

I have a question related to this. I would like to isolate mtDNA from blood cells. We only have a limited amount of frozen blood cells (20x10^6) and would like to have pure mtDNA (for LC-MS measurements of DNA methylation). Which method do you suggest?

So far we tested different cell lysis methods followed by centrifugation, but we ended up with quite a lot of contamination with nuclear DNA. We are planning to use Dnase 1, how much DNase 1 (mg/ml) should we add to 30x10^6 cells? (We are a bit afraid to use a gradient because we have the feeling you easily loose material/mitochondria?)

Somebody also suggested loading the (total) DNA on gel and cut out the mtDNA band, is that a possible solution?

Mitochondrial work is completely new for our lab so all suggestions are welcome!

I used the mtDNA extraction kit from WAKO to extract from cell culture and got good results in the past. If you want more putiry, extract mtDNA with this method, then run an agarose gel with EtBR, incise the 16Kb band and purify using Qiagen kit. You should have pure mtDNA good for sequencing.

DNase1 is an endo-nuclease that digests circular DNA such as mtDNA. You will lose mtDNA. I would suggest Plasmid-Safe™ ATP-Dependent DNase (Epicentre) that is an exo-nuclease and only digests linear DNA (nuclear DNA) and preserve circular DNA (mtDNA). The enzyme comes with protocol that works very well.

Because it is circular, mtDNA migrates differently from linear 16-kb DNA marker, and gel-purification may not work.

If it's human blood mtDNA is extracted from, NheI or BmaHI will linearize it before electrophoresis.It will anyway probably need to be hydrolysed if it's for LC-MS analysis. If not it can be linearized again.

Hi Puck.

I´m afraid that you will never get enough mtDNA to see it on gel from your samples. Majority of the volume of a white blood cell is nucleus, so with small amounts you will have major problems with getting rid of nDNA. You can try Dr. Tanaka´s suggestion, but it might be almost impossible to verify what you actually measure with LC-MS. I would start by setting up the method on good quality mtDNA, which you´d have in high amounts, such as from rat liver.

Cheers,

Jaakko

We have tried all methods based on centrifugation, but there is allways nuclear DNA contamination, except with ClCs gradient ultracentrifugation. We did not tried gel band purification. That could work, yes, with 0.5% agarose gel. Good luck!

I agree with Jaakko's comment about practicing, but the problem is that getting the method to work with liver is not a guarantee at all that it will work with blood cells, it's a fairly different tissue...Maybe, trying to practice with suspension cell culture would be a closer match.

Puck has 20,000,000 cells. Even if we suppose the mtDNA copy number for blood cell is low, say just 10 copie per cell, he will have 200,000,000 copies of mtDNA...which should be enough to see on a gel even with a meagre 10% yield.

Whatever you decide, please let us know how you managed.

Bye

Thank you very much for all suggestions! We are optimizing the mtDNA isolation first with HEK293T cells before using blood samples. Tanaka's exonuclease sounds like a good idea. I red that benzonase might also work? But this is an endonuclease so is not specific for linear DNA, right?

We will also try loading it on agarose gel.

2+E8 mtDNA molecules equals 3.3E-16 moles, totaling about 3.6ng of 16569 bp double stranded DNA. Total blood copy number is probably around 300 mtDNA/nDNA, but considering the yield issues, I guess the above estimate is reasonable.

My earlier point was that it will be difficult to say how much you will measure contaminating nDNA. Benzonase is used to clear up contaminating DNA when your mitochondria are still intact (which I doubt in the case of frozen samples). In blood samples the leucocytes are the ones that are meaningful for mtDNA, and they consist of mostly nucleus and are hard to break.

You will not get pure mtDNA from your samples by any method. With clean enough mtDNA (from your HEK or preferably then Jurkat cells) you could do titrations with total DNA to see when does it begin to matter in your application and also compare it to isolating mtDNA from amounts of HEKs similar to your blood samples. This way you could give the reviewers estimates how reliably your method works with the actual samples. nDNA has much higher levels of methylation, so you might be able to some maths. I´m sorry but I don´t see any other way.

Joachim is in the same institute with you, so he´s probably happy to help. :-)

Cheers,

Jaakko

Sucrose gradient centrifugation is the best method for mitochondrial isolation which then can be used for mtDNA isolation. For many of the PCR based methods one need not be bothered about pure mtDNA isolation as the mitochondrial gene-specific primers can solve the problem.

High through put for Mitochondrial isolation can be achieved via Sucrose gradient centrifugation. This is also very useful in mtDNA isolation.

Just be careful with the primers for not amplifying pseudogenes. Don't forget to blast the sequences

There are several steps that need to be carried out when isolating mitochondrial DNA from tissue sample:

1) Tissue homogenization

Tissue homogenization represents the first step in the mitochondrial DNA (mtDNA) isolation workflow when starting with tissues e.g., muscle, heart, liver, brain, or kidney. To obtain sufficient amount of mtDNA for downstream analysis sufficient amount of mitochondria need to be prepared. High quality mitochondria preparation in terms of purity and yield is again dependent on efficient tissue homogenization. Homogenized tissue can be prepared using the Mitochondria Extraction Kit - Tissue in combination with gentleMACS Dissociators from Miltenyi Biotec. The combination of validated kit and instrument enables optimal tissue homogenates and provide the ideal basis for subsequent isolation of intact, functional mitochondria at high yield.

2) Mitochondria isolation

Mitochondria isolation can be performed by differential centrifugation or by a novel magnetic bead-based method. In this case the magnetic beads are conjugated to an anti-TOM22 antibody and allows gentle and specific mitochondria isolation using columns mounted in a magnetic field. The magnetic bead-based method gives better purity and higher mitochondria yield than differential centrifugation. The bead-based method is available in a kit format from Miltenyi Biotec which contains magnetic beads and all necessary buffers for lysis, isolation and storage as well as columns.

3) Mitochondrial DNA isolation

Mitochondrial DNA isolation can finally be carried out using commercially available DNA isolation kits.

Commercial kits may not work uniformly for different organisms or tissues. This need to be standardized while using such kits

I am now in contact with Joachim for the mtDNA isolation.

I have one more question: does any of you know whether there is an array available to measure gene expression of all mitochondira-encoded genes at once? I only see a hMitChip3 array which includes the 37 mtDNA genes, but also many other nuclear genes.... Ofcourse we can develop all qPCRs ourselves (or order from taqman), but if there is an array available this will be much more convenient (and hopefully cheaper).

Hi to all!

I need to isolate mtDNA from fishes, and have few questions.

First, if there is anybody with experience in fish mtDNA isolation I will appreciate to be in contact.

Since we need to isolate many samples of mtDNA, I'm trying to find any "high- throughput protocol" . The first step of homogeneization is the limiting one. ... is there any suggestion ?

Is there any influence of tissue preservation and stoarge ( ethanol,DMSO, -20, -80) on the quality and yield of mitochondrial DNA isolation and in the purity?

I need to get rid of as much as possible genomic DNA, ... Hisashi Tanaka has mentioned the use of exonuclease to degrade linear DNA, when do you add this step?

Thanks for the help!

Tamara

Hi Tamara,

Have you considered the use of CTAB and proteinase K for digestion? It´s quite simple and time efficient when handling large amount of samples. No need to say that the fresher the samples are the better. If you have the chance to choose from different preserved tissues I would select -80 (nice conservation conditions and less PCR problems ahead), if you have alcohol preserved tissue consider washing alcohol previous to DNA extraction.

Good luck.

We have been doing this sort of work. We use the Seahorse plate reader for looking at OCR and ECAR in freshly isolated mitochondrial from brain hippocampus. Then we perform Western blots on mitochondrial ETC proteins using the mitoprofile kit from Abcam. I dont think we are seeing the same levels of so called contamination as you are.

Thanks Eduardo!!!!!!

Would CTAB work for blood as well?

I think tek buffer method will help to isolate mitochondrial dna isolation

I need to mtDNA for measuring epigenetic marks on the DNA. I cannot use the PCR based method since it will alter the marks on the mtDNA. I found the abcam kit. Does anyone have any personal experience of using it?

http://www.abcam.com/mitochondrial-dna-isolation-kit-ab65321.html

Any help would be appreciated.

We have recently published one paper on preservation and extraction of DNA from Zoanthids. Paper is available as download - full text on my researchgate account.

Hi!

Maybe you would like to read that?

Wilber Quispe-Tintaya et al, Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing.Biotechniques. 2013 Sep; 55(3): 133–136.

In the paper is present a fast and cost-effective two-step protocol for isolation of mtDNA from mammalian cells that is suitable for NGS analysis: (i) extraction of an mtDNA-enriched fraction using a common plasmid miniprep kit and (ii) further purification of mtDNA using the Agencourt AMPure XP system. This method is capable of an up to ~2000-fold enrichment of mtDNA, is superior to commercial kits, and costs a fraction of the price.