Hi,
I'm performing a differential analysis using limma between control samples and samples from two related diseases from approximately 100 patients in each group. The experiment is simple in that we are trying to find transcripts differentially expressed in each disease compared to control samples.
The diseases tend to occur more frequently at different body sites on the skin, so in the design of the experiment, we have collected two samples for each control sample. In some cases we have collected 3 control samples and all of the diseased samples are from independent patients. The numbers of samples look like this.
arm back leg scalp CTRL 14 99 100 0 D1 37 2 44 0 D2 10 90 1 19
I'm currently using limma whilst blocking for body site with the contrasts D1-CTRL and D2-CTRL however I'm thinking that treating replicate control samples from different body sites as independent samples is incorrect and that I am underestimating the variance in the control group. Could anyone suggest the best approach to deal with this? Would adding the replicates as a random effect using duplicateCorrelation() be appropriate in this instance? Ideally I’d like to keep all control samples in the design so that I'm comparing against a common control baseline.
Cheers,
Owen
It isn't clear what you mean when you say "We have collected two samples for each control sample." It seems that you are using the word "sample" for two different things. Do you mean that you took two tissue samples from each part of the body for each healthy individual? If not, could you explain more explicitly what you have done?
Apologies, that was a very confusing mistake. I meant that we collected two samples (one at each body site) for each healthy individual.
OK, that's clear now.