[NHCOLL-L:1456] Formalin & DNA

[Ross MacCulloch]

Tonya:

Try as we might, we have never been able to extract good long chunks of DNA from formalin-fixed specimens.  The best that we've been able to do is to obtain relatively short segments, nothing like we get from fresh, frozen or ethanol-fixed tissue.  Nonetheless, I'd be very interested in finding out about anyone who did have success with formalin-fixed tissue, and their protocols.  Could you please cite those papers here?

We have a large frozen tissue collection, but lately we've been fixing tissue in 95% ethanol, rather than freezing it, for future molecular work.  Frozen tissue is required for protein electrophoresis, but we seldom use that technique any more, preferring DNA sequencing. Ultracold freezers are expensive. Good sequences can be obtained from ethanol-fixed tissue, and when in the field it's a lot easier to put tissue into a 1.5 ml tube with some 95% ethanol than it is to transport a tank of liquid nitrogen for tissue freezing. Carrying a litre of ETOH rather than a 10+ kg LN2 tank makes  big difference on a field trip!

Alternatively, you can fix the entire animal in ethanol (some European collections did this historically), and remove tissue later as needed. However, there are drawbacks to this: 1) formalin fixation produces better, harder, specimens than does alcohol fixation and 2) if you want tissue at a later date, you have to remove and dissect the specimen.  Better to remove a bit of tissue (we take liver) when the animal is first collected, put the tissue into ethanol, and fix the specimen in formalin.

We store ethanol-fixed tissue tubes in a regular freezer (-10 to -20 C).  Our primary reason for this is to reduce ethanol evaporation.  We have also stored tubes at room temperature for extended periods of time with no noticable degradation of DNA.  It should be noted, however, that since ethanol preservation of tissue is relatively new technique, there are no data on the effects of temperature on long-term storage of tissue in ethanol.

Organization of tissue collections is another issue, which has been debated on Gencoll-l.


So in summary:
1) Extracting DNA from formalin-fixed tissue is problematic
2) Store tissue samples in 95% ethanol rather than freezing them in a ultracold. However, if you plan to do protein electrophoresis, you must store tissue, without alcohol treatment, in an ultracold freezer.
3) Fix the specimens in formalin.

Best wishes

Ross D. MacCulloch
Assistant Curator - Herpetology
Centre for Biodiversity and Conservation Biology
Royal Ontario Museum
100 Queen's Park
Toronto
Ontario M5S 2C6