Dear Colleagues:

A couple weeks ago I asked for advice concerning tissue storage and DNA
preservation. Below I include the original posting and the responses I
received (unless otherwise explicitly noted not to).

I was really amazed at the response from the group on this topic and
also the variety of answers. I leave it up to you to decide what to
make of the recommendations.

Thanks to everyone who sent in comments!

Rick

Original post:

I would like to ask for any advice on long term storage and DNA
extraction of/from mammalian tissue (rodent heart, lung, liver, spleen,
muscle, etc.) samples. We have the experience that storing our samples
at 4degrees in 70% ethanol results in a high amount of DNA degradation
after even one year. Is it recommended to extract all the DNA from the
tissues while they are fresh and keep it all in solution at -80 and do
away with tissue storage all together or are there any preferred
methods of tissue storage (with respect to DNA preservation). Does
anyone simply freeze their tissue?



======


Joshua R. Whorley
At the Burke Museum we simply freeze our tissues at -80. We have good
success extracting high quality DNA years after collection.

Mark Schultz
Yes, I just freeze my tissues. 70% EtOH works well too, but an acid
water content may hydrolyse the DNA. Apparently, 95% EtOH is better but
100% is not optimal as it contains Benzine to strip the final 5% water
content from the Ethanol. Once the DNA is extracted, TE buffer is good
for long term storage, as is water at neutral pH and freezing (we do it
at -18 degC). Repeated freeze-thawing also causes DNA fragmentation.
Some citations that might be useful, although they do refer to
invertebrates:

VINK, C. J., THOMAS, S. M., PAQUIN, P., HAYASHI, C. Y. & HEDIN, M.
(2005)
The effects of preservatives and temperatures on arachnid DNA.
Invertebrate
Systematics, 19, 99-104.

DEAN, M. D. & BALLARD, J. W. O. (2001) Factors affecting
mitochondrial DNA
quality from museum preserved Drosophila simulans. Entomologia
Experimentalis Et Applicata, 98, 279-283.

J. Albert Vallunen
We've had similar problems in our lab with fish tissues. 5 to 10 year
old ethanol-stored samples have degraded virtually all of their DNA. It
seems that ethanol concentration has also a role in this: 70% ethanol
seems to be too diluted to preserve the tissue, especially if the piece
is big and moist to begin with. On the other hand, using 100 proof EtOH
fixes the tissue too much pretty soon. Therefore using ethanol which
is 90-96% might yield better results (but we'll have to wait for 5
years to see if it actually works). 

As a solution to all this we are also thinking about extracting all of
the DNA in one go and then storing it in -80. But because extracting
DNA from all of our thousands of samples is a huge task I'd be
interested in hearing any (hopefully different) solutions you receive.


Gregory Maes
we never keep tissue on 70 % ethanol for DNA analysis. It indeed simply
degrades very fast (only used for morphological sample preservation) !
It should be around 95 % not less ! This gives very good preservation
conditions, even more when put in the fridge of -20 °C. 

However, it can still degrade and the best is indeed to purify at least
once the tissue and store high quality DNA in -80 °C (in TE buffer or
ddH2O) or you coudl try to store it on the Whatman FTA papers used in
forensics. They seem to have a good yield afterwards, but you should
test this first.


Francois Pompanon
for frog tissue samples we saw that preservation in silica gel was
better than in ethanol. As in your case ethanol-preserved samples
showed degradation that did not allowed AFLP fingerprinting. However I
think that the best solution is to extract the DNA as soon as possible
after sampling.


B. L. Cohen
I doubt if you will find any widely applicable consensus.

I have some brachiopod and phoronid DNAs that have been stored as
ethanol pellets under 70% at 4C for almost 20 years and when
redissolved and cleaned up give amplifiable 1kb+ products. Similarly
some, but not all, brachiopod tissues at 4C
have been usable after ~20 yrs. But being scared I keep most at -20,
also under ethanol. I ran an enquiry recently about buffering the
ethanol (perhaps important for me because of calcite shells) and
learned from one or two replies, especially from Blair Hedges
(tetrapods mainly), that some people do buffer 70% for long-term tissue
storage, e.g. dilute the ethanol with 1 x TE. That seems like a sound
practice. There is one literature reference suggesting that isopropanol
is NOT a good idea. My experience with it is mixed, perhaps because
industrial
grade, which I had to use in New Zealand, is contaminated with metals.


Joel Anderson
I would recommend freezing two tissue samples per specimen, in
cryotubes at -80, without any solution or ethanol (i.e. freeze only the
tissue). In the past, I have kept two samples; one sample is a
"working" stock of tissue, that is used for all extractions (keep
thawing to a minimum), and the second is a "backup" tissue which I do
not thaw unless my working stock has gone bad.
Further, if you have the opportunity, freezing tissues in the field
(using a portable liquid nitrogen container) is ideal. Otherwise,
you may store tissues temporarily in ethanol, and then pat them dry
before freezing them. Whatever the case, if you
are freezing the tissues at -80, there is no need for the ethanol.

I have done this with rodent tissues (murids and others from South
Africa) and was able to amplify large (~3000 bp) gene regions after
spending 6 weeks in the field.


Kevin J. Roe:
I have frozen tissue at -80 with and without using a buffer (buffered
Tris, pH 8.0) and haven't noticed any significant difference. The
critical difference appears to be when the samples are thawed, I think
the buffered samples fare better after thawing and re-freezing.


Liliana M. Davalos

A
great book on this subject, and a somewhat related reference attached



http://www.kewbooks.com/asps/ShowDetails.asp?idT4



Martin Wiemers
why don't you use 99% ethanol? This should be much better than 70%
ethanol. If tissue contains a lot of water I would change the ethanol
after a while to keep the ethanol concentration as high as possible. My
experience is mostly from insects, but mammal tissue should not be so
different. For long term storage I would put the ethanol samples in a
normal freezer. I don't think -80°C storage is useful unless you want
to store fresh tissue. Even then, a simple freezer (-18°C) will do, at
least for a year or two, and the tissue is definitely fine for DNA
extraction and PCR. In this case you don't need ethanol, but in case of
power interruption or freezer fault you might loose the samples wheras
ethanol storage is safe in such cases.


Isabelle Delisle
I have worked with samples preserved in 95% ethanol at -80 as well as
tissue frozen directly at -80. I never had a problem extracting DNA
from them, even for samples approx. 10 yrs old. 


Tameika-Nicole Armstrong
We always stored honey bee larvae for long term storage at -80C in 30%
glycerol


Melissa A. Fleming
Yes, just freeze it! If you have to have it in ethanol in the field
(rather than frozen in liquid nitrogen, which is better), then freeze it
when you get back to the lab. Even a -20 is better than 4 degrees, but
your minus 80 would be perfect. I try to just freeze all my samples, but
I have a few that arrived in ethanol and they are in a -80 too. I'm not
sure whether frozen in 'air' is better than frozen in ethanol, however.
But a quick search on www.google.com using the words "tissue, storage,
freezing, ethanol" brought up a whole lot of interesting studies
comparing the efficacies of different storage methods. I don't have time
to look at them all now myself (but will go back later), but thought you
would want to know.


Jeff Markert
I think it comes down to a need to do some long-term experiments with
tissue preservation. Different tissues from
different animals might preserve differently.

As you mention, DNA will be quite stable, however you do need to make
sure that it doesn't evaporate. If you use
eppendorf (flip top) tubes, you must put parafilm around the tops to
prevent this.

For long-term storage of tissue, you really
need to experiment a bit. I've found that for small crustaceans, 100%
ethanol works quite well compared to DMSO/Salt. On
the other hand, I've had bad yields from fin clips stored in ethanol
for ~1 year (similar to your mammal findings),´but have had excellent
long term (4+ years) yields using DMSO/Salt. You
might also consider replacing the alcohol in your samples periodically.
I think it absorbs water over time. And you can
probably start with 100% rather than 70%. Avoid
using denatured if at all possible.

Hope that helps a bit. If possible, you might
consider storing half of your new samples each
way until you can determine which works best.
 

Diana
Wolf:

i
believe that when dna is dry, it forms an alpha structure, which is
much more
stable than the beta structure it forms when dissolved in a water-based
solution. Therefore, I think that DNA is most stable when dry and cold.
Is it feasible to
dry your samples, perhaps in a speed vac or freeze dryer? 

With plant tissue, I have dried it and stored it at room temperature
for up to
a year and had no problems. For best storage, I dry it and freeze it at
-80


Steve
Smith:

In
response to your query I can offer some advice for storing DNA after
extraction
which I believe to be the best way to ensure high yields in the
long-term. I
did a long-term experiment on DNA preservation while working at the Max
Planck
Institute in Leipzig and published the results in the Journal of
Forensic
Science.Please find a reprint attached to this email and note the
erratum
which corrects the Sigma catalogue number for trehalose. Basically we
found
that storage at -80 in the presence of trehalose preserved thehighest
quantity and quality of DNA but that similar results were also obtained
for DNA
stored dry in the presence of trehalose.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=search&term=Optimal+Storage+Conditions+for+Highly+Dilute


Mariana
Morando:

You
have to use 100% ethanol, after 24-48 hs, you have to change ethanol,
so you
extract the water the tissues liberated into the ethanol (thus diluting
it!).
Of course you have high degradation of DNA using 70%, it has a lot of
water !!!
the worst venemon for DNA. I extracted DNA from 6 years old tissues,
completed
dried out (thus no water), or in 100% ethanol, and they are perfect!! I
even
amplified nuclear genes out ot them. And most of the time they were at
room
temperature all these years. NO WATER to DNA, and you'll be OK. Keep
your
tissues! just use dpure ethanol! even let them at room temperature, DNA
is
highly resistant to temperature.


Christopher N. Balakrishnan:
I
think storage at 4 degrees might be the problem. 

I have stored tissue samples in a DMSO buffer at -80 (or even -20) for
years. 

It is also fine to just store frozen tissues at -80 with no buffer. 

If you are collecting samples in the field, the best method is to snap
freeze
samples in liquid nitrogen. It just depends on whether you can carry
around a
liquid nitrogen tank... 

Another good option would be to use RNAlater. This is great, because it
will
preserve RNA and DNA, but is a quite expensive. 

Ethanol should also be fine though, as long as you store samples in a
freezer as soon as possible. One problem I have had with ethanol, is
that is
some countries, the only ethanol available is "denatured" ethanol.
Storage in denatured ethanol will definitely cause your DNA degrade
quickly.
I've had this problem with ethanol purchased from pharmacies in
Cameroon. And
I've heard of others having this problem.

Carol
Mariani:
Mammalian
tissues stored at
-80 in 70% ETHANOL should be stable for many, many years. 
DNA
Extractions kept at -80 should also be stable for just as long. 



-- 
Rick J Scavetta
Abteilung fuer Evolutionsgenetik
Institut fuer Genetik
Universitaet zu Koeln
Zuelpicherstrasse 47
50674 Koeln - Germany
Tel: ++49 221 470 3402
Fax: ++49 221 470 5975
rscavett@uni-koeln.de