1b. The experimental unit
What to write
Describe the experimental unit (e.g., a single animal, litter, or cage of animals).
Explanation
Within a design, biological and technical factors will often be organised hierarchically, such as cells within animals and mitochondria within cells, or cages within rooms and animals within cages. Such hierarchies can make determining the sample size difficult (is it the number of animals, cells, or mitochondria?). The sample size is the number of experimental units per group. The experimental unit is defined as the biological entity subjected to an intervention independently of all other units, such that it is possible to assign any two experimental units to different treatment groups. It is also sometimes called the unit of randomisation. In addition, the experimental units should not influence each other on the outcomes that are measured.
Commonly, the experimental unit is the individual animal, each independently allocated to a treatment group (e.g., a drug administered by injection). However, the experimental unit may be the cage or the litter (e.g., a diet administered to a whole cage, or a treatment administered to a dam and investigated in her pups), or it could be part of the animal (e.g., different drug treatments applied topically to distinct body regions of the same animal). Animals may also serve as their own controls, receiving different treatments separated by washout periods; here, the experimental unit is an animal for a period of time. There may also be multiple experimental units in a single experiment, such as when a treatment is given to a pregnant dam and then the weaned pups are allocated to different diets1. See2–4 for further guidance on identifying experimental units.
Conflating experimental units with subsamples or repeated measurements can lead to artificial inflation of the sample size. For example, measurements from 50 individual cells from a single mouse represent n = 1 when the experimental unit is the mouse. The 50 measurements are subsamples and provide an estimate of measurement error and so should be averaged or used in a nested analysis. Reporting n = 50 in this case is an example of pseudoreplication5. It underestimates the true variability in a study, which can lead to false positives and invalidate the analysis and resulting conclusions5,6. If, however, each cell taken from the mouse is then randomly allocated to different treatments and assessed individually, the cell might be regarded as the experimental unit.
Clearly indicate the experimental unit for each experiment so that the sample sizes and statistical analyses can be properly evaluated.
Examples
‘The present study used the tissues collected at E15.5 from dams fed the 1X choline and 4X choline diets (n = 3 dams per group, per fetal sex; total n = 12 dams). To ensure statistical independence, only one placenta (either male or female) from each dam was used for each experiment. Each placenta, therefore, was considered to be an experimental unit’7.
‘We have used data collected from high-throughput phenotyping, which is based on a pipeline concept where a mouse is characterized by a series of standardized and validated tests underpinned by standard operating procedures (SOPs)…. The individual mouse was considered the experimental unit within the studies’8.
‘Fish were divided in two groups according to weight (0.7–1.2 g and 1.3–1.7 g) and randomly stocked (at a density of 15 fish per experimental unit) in 24 plastic tanks holding 60 L of water’9.
‘In the study, n refers to number of animals, with five acquisitions from each corticostriatal slice, with a maximum of three slices obtained from each experimental animal used for each protocol (six animals each group)’10.
Training
The UK EQUATOR Centre runs training on how to write using reporting guidelines.
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