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PLoS Biol. 2018 Apr 4;16(4):e2005282. doi: 10.1371/journal.pbio.2005282. eCollection 2018 Apr.

What exactly is 'N' in cell culture and animal experiments?

Author information

Quantitative Biology, Discovery Sciences, AstraZeneca, Cambridge, United Kingdom.
School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom.
MRC Integrative Epidemiology Unit, School of Experimental Psychology, University of Bristol, Bristol, United Kingdom.


Biologists determine experimental effects by perturbing biological entities or units. When done appropriately, independent replication of the entity-intervention pair contributes to the sample size (N) and forms the basis of statistical inference. If the wrong entity-intervention pair is chosen, an experiment cannot address the question of interest. We surveyed a random sample of published animal experiments from 2011 to 2016 where interventions were applied to parents and effects examined in the offspring, as regulatory authorities provide clear guidelines on replication with such designs. We found that only 22% of studies (95% CI = 17%-29%) replicated the correct entity-intervention pair and thus made valid statistical inferences. Nearly half of the studies (46%, 95% CI = 38%-53%) had pseudoreplication while 32% (95% CI = 26%-39%) provided insufficient information to make a judgement. Pseudoreplication artificially inflates the sample size, and thus the evidence for a scientific claim, resulting in false positives. We argue that distinguishing between biological units, experimental units, and observational units clarifies where replication should occur, describe the criteria for genuine replication, and provide concrete examples of in vitro, ex vivo, and in vivo experimental designs.

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