Using the limma package I compared the mRNA expression profiles between small groups (N=5) of isogenic mice (balb/c) for different conditions (Agilent one-color microarrays). I could observe strong p-value inflation (as measured by lambda) in most comparisons despite the FDR correction. One factor known from genomewide association (GWA) studies to cause p-value inflation is population stratification, such as relatedness among individuals. Whatever the comparisons, all the mice are supposed to have the same genetic background. Therefore, I have several questions:
1) Do I need to adapt sepcific limma functions to take this study design into account?
2) Because one-color microarrays were used for this experiment, I have been told that within array normalization may not be feasible. Until now this has not been applied to the dataset. What do you think?
3) Do you think that additional packages such as BACON may be necessary to fix this issue?
Thank you very much for your help