Dear all,
I have a project to analyze where the researchers used 3 different patients (human primary cells) and applied different stimuli on each of them. Here is a short description of the experiment:
Patient A: no stimulus, stimulus 1, stimuli 1+2
Patient B: no stimulus, stimulus 1, stimuli 1+2
Patient C: no stimulus, stimulus 1, stimuli 1+2
Then, I have biological triplicates for each condition (no stimulus, stimulus 1 and stimuli 1+2) but by qPCR they noticed the basal level of some of their genes or interest were no similar but after the different stimuli, when they look at the fold-changes (normalized by the no stimulus condition), they get similar fold-changes between the patients.
Now, I have to analyze mRNAseq of these patients in each condition and I wanted to do a Differential Expression analysis (with DESeq) but I have a doubt. If I take the 3 patients in one condition, the raw counts will be different for a gene (actually even in RPKM)... Then, I am not sure I can really consider my 3 patients as triplicates.
Or, should I do DE analysis of each patient separatly considering I do not have replicates and then compare the obtained fold-changes?
Or even better, is there any DE package (I love R) which may take into consideration the patients?
Sylvain
I have a project to analyze where the researchers used 3 different patients (human primary cells) and applied different stimuli on each of them. Here is a short description of the experiment:
Patient A: no stimulus, stimulus 1, stimuli 1+2
Patient B: no stimulus, stimulus 1, stimuli 1+2
Patient C: no stimulus, stimulus 1, stimuli 1+2
Then, I have biological triplicates for each condition (no stimulus, stimulus 1 and stimuli 1+2) but by qPCR they noticed the basal level of some of their genes or interest were no similar but after the different stimuli, when they look at the fold-changes (normalized by the no stimulus condition), they get similar fold-changes between the patients.
Now, I have to analyze mRNAseq of these patients in each condition and I wanted to do a Differential Expression analysis (with DESeq) but I have a doubt. If I take the 3 patients in one condition, the raw counts will be different for a gene (actually even in RPKM)... Then, I am not sure I can really consider my 3 patients as triplicates.
Or, should I do DE analysis of each patient separatly considering I do not have replicates and then compare the obtained fold-changes?
Or even better, is there any DE package (I love R) which may take into consideration the patients?
Sylvain
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