I have two bacterial strains - one is wild type and the other has one gene deleted. In my experiment, I calculate some observable characteristic (which is continuous) for both of these strains. I repeat this experiment 5 times. Now I want to see if there is a difference between the two strains, so I perform a t-test (assuming the data is normally distributed for now). My question is - should I perform a paired t-test or unpaired? I am a bit confused about it since I feel I have some rationale for doing it either ways -

Why I think its paired : in every replicate, I compare the same two strains and I repeat this 5 times

Why I think its unpaired : Its not like I am subjecting the same strain to two different conditions (which often happens in paired tests). Rather, I have two different strains being subjected to the same condition.

Any help would be appreciated!

Cross posted at Biostars.org

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    $\begingroup$ I suppose you, for each strain, have five distinct bacterial samples from that strain. Why do you pair them off? Is each replicate pair done under different conditions, for some reason? If so, maybe paired. Otherwise, you seem to have just two independent samples which just happens to be of same size and organized in pair. That woud be an unpaired test. Can you clarify? $\endgroup$ May 27, 2021 at 1:41

1 Answer 1


The question you need to address is whether there are substantial systematic differences among experiments, e.g., from day to day. If so, then there might be an advantage to pairing.

The t statistic is the ratio of the difference in means to the standard error of the difference. If there are systematic differences among experiments, then pairing can reduce the standard error of the difference and give you a higher t statistic than with unpaired data.

The tradeoff is that the paired t-test has fewer degrees of freedom. The greater the number of degrees of freedom, the lower the value of the t statistic you need to document significance. With 5 pairs of wild-type/mutant samples, a paired t-test would only have 4 degrees of freedom. An unpaired t-test on 5 per group and 2 groups would have 8 degrees of freedom.

This page and its links go into more technical detail.


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