With reference to reference genes – or, you’re doing it WRONG!

Allegorically, science is sometimes a meandering river. Photo by Flickr user Tim Haynes, used under a CC BY-NC-ND 2.0 license (And what a great shot it is!)

A lab is an organic thing that evolves over time. People come and go, grad students graduate and the research shifts; sometimes slowly like a meandering river, other times fast like a ricochet from a bullet. Three years ago our lab started a new project, aimed at understanding the role of innate immunity of ducks for fending off pathogens. Contrary to the adaptive branch of the immune system (T-cells and B-cells, antibodies and the like), the innate immune part is still mostly a black box for wildlife diseases. It contains a bunch of forces, from patrolling cells, eating bugs, to specialized molecules that stamp little holes in bacterial membranes or that ring the alarm bell to recruit other diverse armed forces. In ten years time ecologists will be all over the place with innate immunity – remember where you read it first!

I love to start new projects! Its great! Everything is possible! You get that pioneering feeling, chartering unknown areas for SCIENCE! Yay! However, after some time a realization dawns that there is a quite a long starting stretch before you can harvest. If you try to pick just the cherries, you run the risk of making uninformed choices, simply because there may be fundamental stuff specific to this new field that you were unaware of when you started. In our case, we are just about to publish some really cool stuff on mallard innate immunity, ranging from the evolution of innate immune genes to the actual responses of these genes upon infection. There is so much awesomness in that project that it could be the sparkle for a whole new lab. But before getting where we are at now, we have spent an eon in the lab optimizing and tinkering with protocols. You see, it is all about the little things, as finding the right primers, or the best way of extracting RNA from tissue samples.

A little while ago, we published a study that was part of this learning curve. What we wanted to do was to look at upregulation of a particular part of the innate immune system. We had done an infection experiment on mallards, testing whether natural infection with influenza A virus initiated increased expression of some target genes in the host. However, in order to be able to really say that a gene is up- or down-regulated, you need to make sure that you normalize your data to genes with stable expression, i.e. genes that do not respond in specific ways to infection.

Doing that exercise, we found that most of the studies previously conducted in our field had used genes that weren’t really stable. This prompted us to take a broader look at the literature to see how things are done, relative to how it should be done. This meta-analysis was published in PLOS ONE two weeks ago.

In short, we show that despite a common approved methodology, researchers still use too few reference genes and in most cases do not make sure that these genes really are stable in their study organism, in the tissue of choice, and under the experimental system under study. Or to quote parts of the abstract:

Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species.

Another way of phrasing it would be: You’re doing it WRONG!

Link to the article:

Chapman, J. & Waldenström, J. 2015. With reference to reference genes: a systematic review of endogenous controls in gene expression studies. PLoS ONE 10(11): e0141853. doi:10.1371/journal.pone.0141853

Head lice – the unwanted gift that keeps on giving

Human head louse, Pediculus humanus, in close-up. [Photo by Flickr user Giles San Martin under a CC BY-SA 2.0 license].

Human head louse in close-up. [Photo by Flickr user Giles San Martin under a CC BY-SA 2.0 license].

There are two telltale signs that your kid’s class is infested with head lice (or in scientific prose Pediculus humanus subspecies capitis). First, and foremost, scout for kids that obsessively scratch the back of their heads – chances are they have a little colony of lice brewing. Second, as many treatment formulas contain oily substances, beware the greasy appearance of post-treatment hair. But most of the time you don’t need to be in doubt, as there is usually a sign on the school door saying ‘there are head lice around’. In my little town, and in Sweden in general, head lice are now a normal occurrence in the younger kids, and an unwelcome gift when the children gets back to school in autumn.

And once a school has them, it seems they are impossible to weed out. Head lice infestations still carry a social stigma, especially among the older generation. Head lice were more or less exterminated in Sweden in the 1950-1980s, and were thought to be restricted to poor people with limited access to sanitation. However, that’s not true anymore (or perhaps never was) – there are head lice climbing around the offspring of lawyers, bankers and…. ahem… professors. One reason for this re-emergence is evolved resistance to the compounds used to treat lice infestation, but likely our ever increasing busy lifestyle is to be blamed too, as effective treatment really only is doable with the painstakingly gruesome louse combing (which most little ones think is torture).

Contrary to most people, I think lice are fascinating critters – at least as long as they stay out of my hair. They are extremely adapted to their niche: the flowing hair of the human skull. Inside this jungle, the lice dart around fast as lightning from one end to the other, usually without us knowing it. However, if you catch one and put it on a flat surface you’ll see that it is unable to move, as the louse’s legs stretches horizontally, adapted to efficiently grab hairs, rather than strolling around. You can also see whether they have fed or not, and from that gather whether a new generation of lice are being produced.

Furthermore, consider the boom and bust dynamics that goes on day after day at the louse population level. Each human head is an isolated patch of habitat, and migration to other patches dependent on the behavior of the host – when we hug each other, swap caps, or in other ways put our hair together – behaviors that differ dependent on age class and gender of the host. For example, in my thinning hair, the poor lice have limited chance of long-term survival (a windswept barren heath land, from a louse perspective), while in my youngest daughters the hair is long and interactions with other suitable hosts frequent. Even so, most colonizations of a patch are likely to involve only a single or a few lice, from which a new generation need to stem. Sexual reproduction is the norm, so either the adult female needs to fertilized before moving to a new host, or she has to have company of a male to start a new colony.

But when the population of lice increase, so does our likelihood to notice them. And once noticed, full war is waged in order to exterminate them. This involves various expensive shampoos, often containing substances that immobilize and suffocate the lice, and, of course, good ol’ fashioned combing. The effect is recurrent population bottlenecks, where numbers of lice go down to very low level before bouncing back. One wonders how they manage to avoid deleterious inbreeding effects.

It isn’t an easy life being louse, loathed and hated by their hosts – but it is the only life they know. Simply, lice are good at being lice, and are unlikely to ever (as in evolutionary times) leave their fitness landscape peak and abandon parasitic life. As for now, they are the unwanted gift that keeps on giving.