What exactly is ‘N’ in cell culture and animal experiments?

Stanley E. Lazic*, Charlie J. Clarke-Williams, Marcus R. Munafò

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

46 Citations (Scopus)
284 Downloads (Pure)


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.

Original languageEnglish
Article numbere2005282
JournalPLoS Biology
Issue number4
Early online date4 Apr 2018
Publication statusPublished - 4 Apr 2018

Structured keywords

  • Brain and Behaviour
  • Tobacco and Alcohol


  • Journal Article

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