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