A few weeks back I asked about the use of propylene glycol (nontoxic, not flammable) to preserve insect tissues for DNA work. Thanks to all those who replied. Below I include a summary of the replies (after the original post) including a summary of my own results using my own focal group - silphid beetles. The short answer is that there were few replies but most were positive about the value of PG. ORIGINAL POST: This paper recent paper on the use of 100% propylene glycol for preservation of spiders for later DNA extraction is exciting because propylene glycol is non-toxic (it's a food additive) and thus can be safely mailed and carried on planes etc. It effectively dehydrates the tissues. The effects of preservatives and temperatures on arachnid DNA Author(s): Vink CJ, Thomas SM, Paquin P, Hayashi CY, Hedin M Source: INVERTEBRATE SYSTEMATICS 19 (2): 99-104 2005 I'd like to know if anyone has additional experience with this method of preservation. The authors used spiders so one might assume the method would work for all arthropods - has anyone used it with insects? I wonder specifically if larger bodied insects might not preserve as well in PG as in ethanol if the ethanol penetrates the tissues to dehydrate them faster than PG. I'd also like to know if propylene glycol is a viable long-term alternative to high-grade ethanol for specimens stored at -80C. Finally, I'm curious if there are any issues with DNA extraction - does the PG need to be thoroughly removed from the specimen for successful extraction? and if so, how is this done? I'll collect replies off-list and summarize them for a later posting. Thanks! -Derek Sikes REPLY 1: Hey Derek If you take a closer look at the paper, you will see that not only spiders were used, but also scorpions. And these were quite large scorpions. That may partly answer you questions. Just keep in mind that these results were for a month only of preservation. The longer term paper in coming along! Cheers Pierre REPLY 2 (in part) I was just going to follow what they did in the paper, that is to transfer the specimens back to ethanol, keep overnight in a refridgerator and then proceed with the extraction the next day as usual. I would normally transfer leg from ethanol to deinoized water, let sit for about 20 minutes then dry out on kimwipe and proceed to extraction. REPLY 3 Hi Derek, Just to update you - I received my spiders in propylene glycol, transferred whole specimens to ethanol overnight at 4 degrees (in fridge). The next day I used 2 legs from each specimen, dried the legs on kimwipes before adding to lysis buffer (Qiagen-DNeasy kit) and extracted DNA as usual. The results from my PCR using both mitochdrial COI primers and those for a single-copy nuclear locus were excellent for all specimens. Thus, it seems at least for spiders, the propylene glycol is not a problem if you transfer to ethanol for 24 hrs and dry out prior to extracting. Of course my specimens were only collected days earlier so it was very good starting material. Anyway, hope this is helpful. Take care, Jessica Garb Jessica E. Garb Post-doctoral Researcher Biology Department 900 University Avenue University of California Riverside, CA 92521 USA Ph: 909-787-7323 Fax: 909-787-4286 REPLY 4 Hello Derek Unfortunately we didn't include the specimens preserved in propylene glycol for our long term (1 year) storage experiment - we didn't expect propylene glycol to work so well. However, I see no reason why propylene glycol wouldn't be fine for long term storage provided they are kept at -20 or -80, which improves long term storage significantly. Propylene glycol does, however, shrivel soft tissue (eg genitalia). The large scorpions we used were quite water-tight but there seemed to be no problem in the propylene glycol penetrating their tissues. We euthanized them by freezing to ensure they they didn't close their oral, anal and respiratory openings. Whenever I preserve any large or water-tight arthropods (eg weevils) I tend to pull off a leg or two and/or dissect/cut it to make sure the preservative reaches all the tissues. Before DNA extraction of propylene glycol preserved specimens we transferred them to 95% EtOH for one day at 4 °C (see Methods, Specimen preservation, 5) - this allowed the specimens to be dried prior to extraction. Hope this of use. cheers Cor Cor Vink Scientist Biocontrol & Biosecurity AgResearch PO Box 60 Lincoln 8152 New Zealand REPLY 5 Dear Derek I did some assays with PG as a fixative several months ago. I used ascidian tissue and three fixation procedures: ethanol, ethanol plus change to PG after 3 days, and PG directly. I extracted and sequenced the samples after ca 4 weeks, and sequenced a fragment of COI. In short, the best results were with the samples fixed in ethanol, the ones fixed in ethanol and afterwards transferred to PG were not so good, but worked perfectly, the samples fixed in PG alone gave very poor amplifications (I didn't try to sequence the latter). I have also kept samples of several small marine invertebrates in PG and in formaldehyde for comparison. The macroscopic aspect of those fixed in PG began to degrade after some months, while those in formaldehyde looked OK. In my opinion, then, PG may not be good for direct fixation or long term storage, but can be very useful if you transfer samples fixed in ethanol to PG for some time (say, for shipment overseas) and then possibly back to ethanol. This will solve the problems we encounter with shipment of alcohol preserved specimens. I hope that, as usual, you post the answers to your query in the list, because I'm interested in hearing more about it. Does anyone have tried with ethylene glicol? Best regards Xavier Turon -- PLEASE NOTE NEW E-MAIL AND UPDATE YOUR ADDRESS BOOK: xturon@ub.edu Xavier Turon Dept. of Animal Biology (Invertebrates) Fac. of Biology Univ. of Barcelona 645, Diagonal Ave 08028 Barcelona MY OWN RESULTS: 5 Nicrophorus adults (Coleoptera: Silphidae) were captured alive, hind legs were removed. Body and hind legs placed in single 15 ml vial of 97% propylene glycol (Fisher Scientific) and kept at ambient (shade) temperature (west of Orlando Florida, 8 April) for 10 days & on plane etc. (This replicates my standard ethanol based protocol - same vial size and often many days without freezer or refrigerator). Followed protocol in paper - 24 hrs in ethanol at 4°C before extraction using Qiagen kit & PCR for COI (first half) and COII - all excellent! Bands as bright as ethanol-preserved samples. This is very encouraging. (Thanks to Gino Nearns for help with the field work and Chandra Venables for lab work) -Derek Sikes Derek S. Sikes, Assistant Professor Division of Zoology Department of Biological Sciences University of Calgary 2500 University Drive NW Calgary, Alberta, Canada, T2N 1N4 dsikes@ucalgary.ca http://homepages.ucalgary.ca/~dsikes/sikes_lab.htm phone: 403-210-9819 FAX: 403-289-9311 "Remember that Truth alone is the matter you are in Search after; and if you have been mistaken, let no Vanity reduce you to persist in your mistake." Henry Baker, London, 1785 Entomological Society of Alberta: http://www.biology.ualberta.ca/courses.hp/esa/esa.htm