Experimental Design – Part 1 – Hypotheses

Yes, the observation is a correlation, the increase in miR expression could be a cause or could be an effect or could be unrelated to the contraction. This hypothesis would work; it’s specific and testable.

This is a reasonable question but it’s not a hypothesis!  A hypothesis is the answer to the question, a statement that your experiment will test

Yes, this is a testable statement. However, I’m not convinced that you could justify treating human wounds on the basis of just the observation in the question, indeed I wouldn’t grant a human or animal ethics license based only upon that observation. However, ultimately, this is the sort of destination that you would want to end up in; a clinical trial comparing treated vs control treatment and comparing repair rates.

This reads like a hypothesis but is it specific and is it testable? You could argue with me that knocking down or overexpressing miRNA21 and doing a RNAseq or proteomics experiment could test this statement. However, you’d need a tight definition of what “master regulator” means. What data would be sufficient for you to reject the hypothesis? If one gene changed would that be enough? Do you need 2, 3, 10? This is specificity that you are looking for in your hypothesis. Being tight at this stage will help you in planning the rest of the experiment particularly in terms of determining groupings, sample sizes and stats tests to use. Note that these questions are designed to reinforce the learning.

Yes, the “manipulation” involved in synchronising the culture is just to provide the control, here you are correlating expression to time after synchronisation. Your design will take into account reasons why you might see correlation even it doesn’t exist (reverse causation, confounding variables) and your stats tests will be based around correlative measures. A hypothesis for this experiment could be “Lamin A expression displays cyclical changes in expression following synchronisation of epithelial cells”

Nope, try again

In this option, you have disrupted the circadian rhythm machinery and then are doing a comparison between the groups. As written, this experiment doesn’t directly test the circadian rhythms, it only asks if the clock protein influence lamin A expression in any way therefore the hypothesis could be something like; “Lamin A expression is decreased in clock protein knockout mice”

I can see why you would pick this, however in this case you actually have changed something. Here by comparing to littermates you have controlled for lots of the variables and therefore the interpretation of the results can be tighter differences in lamin A expression here are linked to the change in the clock. Click the other answer for the hypothesis

People in the remote tribe might have distinct diet than the control group. Diet would influence bone and muscle growth, density, repair, development etc

Differences in behaviour between the tribe and control group. For example, if the tribes people rely on hunting and gathering type behaviour and the comparators have different professions, then adaptations to gait may not be shoes but rather what the people do in terms of how much they walk, run etc, how often, how far etc

Genetics. If the tribe truly is remote then likely they will have a certain degree of genetic isolation raising the possibility genetic or epigenetic differences that influence gait, muscle, bone growth etc

Terrain/landscape if the tribe live in different underfoot conditions from the comparators then there would be potential confounder of the different stresses put upon the tissues due to eg up and down hills vs on the flat

Cultured cells. Asking this question in an animal model would be possible with the appropriate lines but the added physiological relevance wouldn’t actually help you answer the question. The counterpoint would be that secretion rate depends on other aspects of the local environment. Unless you had a specific reason to say why you thought this was the case then, at very least, you should do the in vitro study first.

3D model,  ex vivo model or animal model? The simplest, cheapest and quickest would be to use cells in culture however here you need a system with fibroblasts and epithelial cells that can effective recapitulate the wound healing response. This is definitely do-able but the question you would ask: it accurate enough” On those basis you could justify using a slightly more involved system. Ex vivo systems are taking something that has been alive and maintaining it outside the body. They can be a good compromise; an animal or human may still provide the sample but it could be spare tissue from an animal that would be euthanised anyway. Setting up your study this way may reduce the animal numbers or reduce the suffering of those that you do have to use. If this was good enough to ask your question, then you are ethically obligated to go down this route. However, wound repair involves immune cell activity in response to epithelial damage. These cells release cytokines which fibroblasts respond to, therefore you may need to use a living animal model to answer this question. The teaching point here, is that these questions are part of the design process and you should be prepared to justify decisions that you have made.

in vitro or ex vivo. At least at first, you want a tightly controlled model system. Here you could test the contact lens efficacy directly on cultured bacteria or take something like an eye from an abattoir and infect it in a lab environment. There is no need, at least at the initial stage, to use an animal. To be clear, before you could go into the clinic, your studies would have to progress to animal and then human trials but the specific hypothesis as written above would be done quicker, cheaper, with fewer animals harmed and with tighter more easily interpreted data in vitro.

Probably human. Two reasons for this answer; firstly, you can collect urine non-invasively from humans so, although you would still need ethical approval, the experiments themselves would have no detrimental effect on the patients. So, why not use the system you care most about (assuming that is human!)? Secondly, if you were to use an experimental animal system you would be inducing the end-stage disease. This means that the animal would have to be sick for extended period of time. It’s hard to justify putting an animal through this level of suffering, so while it would be possible to test your hypothesis this way, and, indeed, it may give you tighter data, the question about whether you should is relevant to the decision you make.