Fig. 3: Randomization of biological replicates across space, time, and batches can reduce experimental bias and reveal interactions between variables.

A Top: An undetected temperature gradient within a lab causes a false positive result. The mutant strain grows more slowly than the wild-type on the cooler side of the lab. Bottom: After randomizing the flasks in space, temperature is no longer confounded with genotype and the mutation is revealed to have no effect on growth. B Top: A chronological confounding factor causes a false negative result. When cells are counted in all of the rich-media replicates first, the poor-media replicates systematically have more time to grow, masking the treatment effect. Other external variables that can change over time include the lab’s temperature or humidity, the organism’s age or circadian status, and researcher fatigue. Bottom: Randomizing the order of measurement eliminates the confounding factor, revealing the treatment effect. C Left: Batch effects exaggerate the similarity between the yellow and green groups and between the purple and blue groups. Right: Randomization of replicates from all four groups across batches leads to a more accurate measurement of the similarities and differences among the groups. Inclusion of positive and negative controls (black and white) can help to detect batch effects. D Randomization is necessary to test for interactions. Left: In hypothetical Experiments 1 and 2, one variable (genotype) is randomized but the other (ampicillin) is not. These observations, separated in time and/or space, cannot be used to conclude that ampicillin influences the effect of the mutation. Right: Both variables (genotype, ampicillin) are randomized and compared within a single experiment. A 2-way ANOVA confirms the interaction and the conclusion that ampicillin influences the mutation’s effect on growth is valid. The two plots displaying the interaction are equivalent and interchangeable; the first highlights that the effect of the mutation is apparent only in the presence of ampicillin, while the second highlights that ampicillin inhibits growth only for the wild-type strain. E–H illustrate other patterns that can only be revealed in a properly randomized experiment. E A low-protein diet reduces respiration rate overall, but that effect is stronger for female than male animals. F Two plant genotypes show a rank change in relative fitness depending on the environment in which they are grown. G Two genes have epistatic effects on a phenotypic trait: a mutation in Gene 1 can either increase or decrease trait values depending on the allele at Gene 2. H This plot shows a lack of interaction between the pathogen strain and the host immunotype, as indicated by the (imaginary) parallel line segments that connect pairs of points. In contrast, the line segments that would connect pairs of points would not be parallel if an interaction were present (see E–G). In H, host immunotype A is more susceptible to both pathogen strains than host immunotype B, and pathogen strain 1 causes more severe disease than strain 2 regardless of host. Image credits: vector art courtesy of NIAID (orbital shaker) and Servier Medical Art (reservoir).