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Analysis checklist: Two-way ANOVA

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Two-way ANOVA, also called two-factor ANOVA, determines how a response is affected by two factors. For example, you might measure a response to three different drugs in both men and women. In this example, drug treatment is one factor and gender is the other. Read elsewhere to learn about choosing a test, and interpreting the results.

Are the populations distributed according to a Gaussian distribution?

Two-way ANOVA assumes that your replicates are sampled from Gaussian distributions. While this assumption is not too important with large samples, it is important with small sample sizes, especially with unequal sample sizes. Prism does not test for violations of this assumption. If you really don't think your data are sampled from a Gaussian distribution (and no transform will make the distribution Gaussian), you should consider performing nonparametric two-way ANOVA. Prism does not offer this test.

ANOVA also assumes that all sets of replicates have the same SD overall, and that any differences between SDs are due to random sampling.

Are the data unmatched?        

Standard two-way ANOVA works by comparing the differences among group means with the pooled standard deviations of the groups. If the data are matched, then you should choose repeated-measures ANOVA instead. If the matching is effective in controlling for experimental variability, repeated-measures ANOVA will be more powerful than regular ANOVA.

Are the “errors” independent?

The term “error” refers to the difference between each value and the mean of all the replicates. The results of two-way ANOVA only make sense when the scatter is random – that whatever factor caused a value to be too high or too low affects only that one value. Prism cannot test this assumption. You must think about the experimental design. For example, the errors are not independent if you have six replicates, but these were obtained from two animals in triplicate. In this case, some factor may cause all values from one animal to be high or low.

Do you really want to compare means?

Two-way ANOVA compares the means. It is possible to have a tiny P value – clear evidence that the population means are different – even if the distributions overlap considerably. In some situations – for example, assessing the usefulness of a diagnostic test – you may be more interested in the overlap of the distributions than in differences between means.

Are there two factors?

One-way ANOVA compares three or more groups defined by one factor. For example, you might compare a control group with a drug treatment group and a group treated with drug plus antagonist. Or you might compare a control group with five different drug treatments. Prism has a separate analysis for one-way ANOVA.

Some experiments involve more than two factors. For example, you might compare three different drugs in men and women at four time points. There are three factors in that experiment: drug treatment, gender and time. These data need to be analyzed by three-way ANOVA, also called three-factor ANOVA. Prism does not perform three-way ANOVA.

Are both factors “fixed” rather than “random”?

Prism performs Type I ANOVA, also known as fixed-effect ANOVA. This tests for differences among the means of the particular groups you have collected data from. Different calculations are needed if you randomly selected groups from an infinite (or at least large) number of possible groups, and want to reach conclusions about differences among ALL the groups, even the ones you didn't include in this experiment.

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