The tissue of the non-fleshy corals such as Acroporidae, and Pocilloporidae family contains much skeletons than the tissues. I had been saw my colleagues working on coral histology and they use 7-10% formic acid to decalcify the skeletons to get the tissue to make histological slide, but is it also safe for preserving genetic materials on corals? If it's not safe, how could I harvest large amount of coral tissue for genetic analyses from non-fleshy corals?
Hi Stevanus,
I'm not too sure about the effect of formic acid on DNA but extraction techniques are so efficient nowadays, even on "non-fleshy corals".
A basic phenol-chloroform/high salt DNA extraction on a piece of coral (skeleton+tissue) will provide enough DNA for a high number of PCR. You simply need to "destroy" the skeleton to release maximum amount of tissue and allow the proteinase to break the cells.
Alternatively you can also air-brush or water-pick your samples to remove the tissue from the skeleton. Someone could argue that these techniques only remove superficially the tissue... but it also depends of your questions.
You could find easily these protocols from the literature. Good luck!
I think DNA analysis does not need much amount of tissues so I guess just water-pick the polyps from the skeleton under a microscope would be the simple technique. I would have done that.
Hi Stevanus,
I found the best method for myself (currently working on extracting DNA from a number of scleractinian families) is to crush the required amount of coral with a mortar and pestle. The skeleton does a good job in lysing the tissue. I usually then store the crushed tissue in a guanidium thiocynate based "CHAOS" buffer for a couple days, followed by a ProK digestion then a standard kit or PCI cleanup... I get very high quality and purity DNA using this method.
If you have coral samples stored in ETOH, you will have to wash the sample thoroughly in distilled water. If you vortex your sample in distilled water, it also helps remove most of the mucus that the coral produces. Mucus can be a problem, especially as it can block silica based flow through tubes and can inhibit PCR.
Good luck!
Dear Stevanus .
There are several tips you can do to extract coral's genomic DNA. These may help you:
1. Manual tissue-picking from polyps (it may work better for corals with larger polyp, such as Favidae and Lobophylliidae) will give you the opportunity to get a much amount of DNA to extract, as well as to make sure you don't pick other symbiotic organism as corals are actually holobiont.
2. Manual grinding may give you an opportunity to get whole genomic DNA easily because the physical forces of grinding and debris friction will help in breaking the cell membrane. Make sure you know the quantity of coral's pieces you need to grind to make sure you don't get an excessive amount of calsium carbonate in your sample. For example, 0.5 cm^2 of Echinopora lamellosa’s piece will give me enough amount of DNA with only a few debris.
3. I recommend you to use spin extraction method than other extraction method. In my case, Qiagen DNeasy blood and tissue always give higher DNA yield than any other extraction kit product, though the price is a bit higher.
4. Longer pre-incubation (overnight, 50-60°C) with Pro-K and lysis buffer will help you to lysis the cell membrane and tissue. This technique works better in my case for increasing DNA yield.
5. To make sure you get pure coral's tissue, try airbrush blasting method by Rosset et al (2019) (https://link.springer.com/article/10.1007/s00338-019-01865-x) to remove possible zooxanthellae. You can also separate coral’s and symbiont’s DNA by using multilevel centrifugation technique as mention by Kenkel & Bay (2018) (https://peerj.com/articles/6047/).
Extraction of coral's DNA may be a bit tough and tricky. Hope this help you.
Regards,
M. I. A. Ghafari
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