I know a lot of biologists who have lists related to their work: lists of birds they have seen, lists of journals they want to publish in, lists of top wildlife spectacles they want to see, lists of species they have studied, lists of their favourite fieldwork sites.
I too have a few lists, and in writing this post I’m thinking of one list in particular: my list of the most disgusting batches of tissue samples I have ever had the misfortune to extract DNA from. It is a short but memorable list, which, as of this week, stands at four entries. And despite the large number of scat DNA samples I have worked with over the years, there are no faeces on that list!
I thought it was going to be a routine afternoon in the lab. I was planning to take subsamples from some roadkilled mammal ear tissues we had been sent. Roadkills can be a valuable non-invasive source of population genetic information for many species. The procedure goes something like this: take a 2mm subsample of tissue, finely chop it with a scalpel and forceps, and transfer the tissue to a tube with lysis buffer to incubate overnight. I had 48 samples to prepare, so was expecting to spend a couple of hours in the lab, then finish the DNA extractions the next morning.
That was the plan… until I opened the first sample tube and very nearly lost my lunch. You see, at some point during the collection, transport and storage process, the samples had started to rot. I won’t describe them in detail, but let’s just say that I quickly reconsidered my approach and took rather longer to process the samples in a less stinky fashion.
Of course the sad part of this story is that, with a little more attention to detail during the sample collection process, the long-term integrity of the samples could have been ensured and my discomfort avoided. Hence this post: a few thoughts on how best to preserve the DNA in tissue collected from roadkilled animals in the field. With a bias towards vertebrates.
First, let’s consider what happens to DNA when an animal dies. This is also relevant to samples such as ear punches or tail snips collected from live animals. It doesn’t take very long at all for decomposition to begin and DNA certainly won’t last forever. Cell walls break down, exposing DNA to the actions of enzymes that can degrade it. Chromosomes become fragmented and eventually DNA can be damaged in other ways that can modify the chemical composition of the nucleotides (a well-known cause of sequencing errors in ancient DNA studies). Moisture, exposure to sunlight, repeated freeze-thaw cycles and excessive heat are also your enemies when you want to preserve DNA.
Obviously the sooner after death you can take your tissue sample the better, and ideally it should be immediately preserved. But which tissues to sample, and how to preserve them? Is it even worth collecting samples from roadkills? Won’t the DNA be too degraded?
Well, the answers to these questions depend to some extent on what you want to do with the DNA. If you want to use it for a genome sequencing project, chromosomal analysis or transcriptomics, you will probably need to sample from a live or very freshly dead animal, sample from multiple different tissue types and organs, and immediately preserve the tissue in liquid nitrogen or a freezer. But for population genetic or phylogenetic studies, which tend to target relatively short gene regions, you can be less picky about the type of tissue you sample and you can get away with some degree of DNA degradation. This is especially true if you are able to focus on genetic markers designed for analysis of trace samples.
With those comments in mind, here are my tips for tissue sample collection from roadkill corpses in the field. NB this list is by no means exhaustive, so if anyone else has tips to share, please do so!
- Sample from extremities such as ears, tail tips, toes and skin as DNA is likely to be better preserved here than in internal organs or muscle tissues from corpses that are several days old. Avoid taking samples from tissues with obvious signs of trauma, decomposition or insect activity, as these are likely to have more DNA degradation. For birds and reptiles you can take blood if the corpse is fresh.
- Store the samples in tubes with a preservative e.g. 95% ethanol, salt-saturated DMSO buffer or RNA Later. But don’t rely on the preservative alone: for long term viability of samples it is still better to get them to a -80C freezer fairly quickly. NB 70% ethanol is not concentrated enough for long-term DNA preservation!
- Don’t try to fit too much tissue into the tube: the preservative buffer needs to be able to soak all the way through the tissue, otherwise it can still rot. Aim for at least twice the volume of buffer as you have sample. Make sure the sample is fully submerged in the preservative.
- Conversely, make sure you take a big enough sample to allow for several DNA extractions, just in case you need to re-extract or share your samples later on. This can be tricky for smaller animals, but if possible I aim for a piece of ear tissue or skin the size of my little fingernail from mammals, or take at least a centimetre from the end of a lizard / rodent tail. For smaller lizards you could sample several toes.
- Make sure that you use tubes that seal properly and that can be frozen. We use 2ml screw-cap tubes with an o-ring in the lid that can be safely stored in our -80 freezers.
- Make sure that the sample from each individual animal gets its own tube – otherwise you will have cross-contamination among samples.
- Make sure that each sample tube is well labelled with a unique sample ID. If you just number the tubes 1-100, you can guarantee you will get confused later. Also make sure the labels won’t come off it the tubes get wet or if you spill ethanol on them. Label both the lid and the tube as a backup, or place a small piece of museum paper, labelled in pencil / suitable ink, inside the tube with the sample. NB if you’re collecting samples as part of a collaborative project with a genetics lab, you may find that they already have a labelling and storage system in place and will send you pre-labelled tubes with buffer, ready to go.
- Collect as much data about the sample as possible: species, sampling date, sex, GPS coordinates and GPS datum used, condition of the carcass (how fresh) and take a photograph just in case there are questions about species ID later on.
- Make sure that you have the correct permits needed to sample from roadkill if that is legally required in your region.
Edited 19/05/2015: I’ve now added a PDF version of these tips for anyone who wants to download an easy reference version for their sampling kit. Please use the link below to access the file. Thanks to Daniela Araya for suggesting this!