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Old 07-10-2017, 02:15 PM   #3
Location: Honolulu, HI

Join Date: Jul 2015
Posts: 32

There's a lot to unpack here, but I can give some advice!

Freezing immediately is great, so long as you have access to those temperatures at the time of sampling. If you can take along, say, some liquid nitrogen (I know people who do this), then you're golden. But the time between sampling/death of the organism and preservation is crucial. This can also depend on what type of tissue you're sampling. Liver gives enormous DNA yields, but tends to degrade much more quickly than, say, muscle tissue.

It's the timing here that is the critical factor. For example, I've sampled shark tissues on 2x consecutive dives where samples collected earlier in the dive stayed with me for ~60 min underwater. It was CA, so the water was chilly, but not that cold. But I tended to notice that samples collected on earlier dives - or even earlier in the dive - tended to give more degraded DNA than the ones that were more immediately preserved/frozen (we'd take the boat back to the lab, and freeze at -80 C). Sometimes the difference was small, even negligible. Other times it really seemed to make a big difference.

If you can't freeze the tissue straight-away (as is the case in most field-situations), a preservative is really the way to go. I saw a paper recently that showed that DMSO gives lower DNA yield than ethanol, but tends to yield higher-MW DNA overall. I'll try to find it.

(There's also RNA-Later, which I tended to use in the field. I used it with mixed success. With RNA-Later, it's important to cool/freeze the tissue gradually... -4 C, to -20 C, to -80 C.)

With regard to preserving in tissue vs. immediate extraction, it follows (from above) that preservation before extraction is still critical. That delay will cause degradation, unless you're literally walking outside, grabbing a live specimen, and walking it back into the lab with you to extract DNA.

Finally, with regard to freezing degrading DNA, what's important is limiting freeze-thaw cycles as much as possible to avoid shearing DNA. At least, that's the common wisdom. But I HAVE heard/read that there's very little evidence (beyond the anecdotal) to suggest that this really does cause significant shearing. Still, best to be cautious.

tl;dr: Unless you can freeze this stuff in the field, put it in a preservative. Some work better than others. I recommend preserving before extracting. Freeze-thaw is a contentious subject, but convention is to avoid too many cycles. An initial freeze most likely won't hurt much at all.
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