Reiss RA, Schwert DP, Ashworth AC. 1995. Field preservation of Coleoptera for molecular genetic analyses. Environmental Entomology 24: 716-719.
These authors tested five preservatives for nuclear and mitochondrial DNA preservation. Carabid beetles collected at Kuujjuarapik, Quebec, from shore-line debris on Hudson Bay were placed into either 95% ethanol, Carnoy fixative (3:1 methanol:acetic acid), DNA isolation buffer (half whole, half homogenized with a pestle), cryotubes immersed in liquid nitrogen followed by storage at -80°C, or glass vials containing tissue paper soaked in ethyl acetate; all treatments except liquid nitrogen were at room temperature. Ethanol is widely used as both a fixative and a preservative, though it does often distort or remove body colouration, and is considered a hazardous material for transport. Ethyl acetate is sometimes used by entomologists to preserve morphology, as it causes less distortion than other techniques such as drying (pinned) or ethanol.
Overall, cryopreservation performed best, producing excellent results when the specimens were subjected to DNA extraction and basic molecular techniques examining both nuclear and mitochondrial DNA. Ethanol also performed well, though specimens maintained in ethanol longer than about 6 weeks showed significant degradation. DNA isolation buffer also performed well, as long as specimens were very thoroughly ground and homogenized; intact specimens did not yield good results.
These authors recommend ethanol for remote field studies where the equipment associated with liquid nitrogen would be very difficult to transport and maintain, and DNA isolation buffer combined with thorough grinding where ethanol cannot be carried due to its hazardous nature.
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2 comments:
Interesting. At SFU, we found that dry specimens yielded DNA that was good enough for mtDNA PCR amplification. We never checked for large fragment integrity. Our observations were not beetle specific, so that may be a source of the difference.
We occasionally had problems if the sample was in ethanol, if stored that way for a "long time", as reported in the paper.
Hello Jim,
Yes, this is the first paper I've read that specifically describes long-term storage of specimens in ethanol and problems with DNA extraction. Most of the people I've talked to about this quite happily store their specimens in high-grade ethanol (e.g. 95%) for months or years, and routinely extract and work with both nuclear and mitochondrial DNA.
Regarding dry insects, my understanding is that dehydrated muscle tissue of the type commonly found in the legs of pinned insects is an excellent source of mtDNA, and probably pretty good for nuclear as well.
Neither method is likely to yield useable cell organelles intact, though. At least as far as I have been able to tell.
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