Hi, I'm a beginner in LIMMA analysis and also microarray data. Hope someone can give me some suggestions.
Here is the frame of my microarray data. There are 40 patients in my study and they draw blood at 4 time points: baseline, 6-month follow-up, 12-month follow-up, and 24-month follow-up. Each patient is classified as a responder or a non-responder.
No Filename ID Response Month 1 D1-1.CEL 1 responder 0 2 D1-2.CEL 1 responder 6 3 D1-3.CEL 1 responder 12 4 D1-4.CEL 1 responder 24 5 D2-1.CEL 2 responder 0 6 D2-2.CEL 2 responder 6 7 D3-1.CEL 3 non-responder 0 8 D3-2.CEL 3 non-responder 6 9 D3-3.CEL 3 non-responder 12 10 D3-4.CEL 3 non-responder 24 11 D4-1.CEL 4 non-responder 0 12 D4-3.CEL 4 non-responder 12
I think my data frame is similar to 9.7 Multi-level Experiments (p.50) in the user guide of LIMMA. (https://bioconductor.org/packages/release/bioc/html/limma.html) Is it correct?
Then I modified the code in the user guide. Below is my code. Based on this data, I want to answer 3 questions.
- Are there differentially expressed genes at baseline between responders and non-responders?
- In 40 patients, Is the expression level of genes changed between baseline and 6-month follow-up?
Is the expression level of genes changed between baseline and 6-month follow-up in responders and poor responders, respectively?
Treat <- factor(paste(data_40$response,data_40$month,sep=".")) design <- model.matrix(~0+Treat) corfit <- duplicateCorrelation(data_40,design,block=data_40$case_id) corfit$consensus fit <- lmFit(data_40,design,block=data_40$case_id,correlation=corfit$consensus) cm <- makeContrasts( res1vsres0ForM0 = Treat1.0-Treat0.0, res1vsres0ForM6 = Treat1.6-Treat0.6, M0vsM6Forres0 = Treat0.6-Treat0.0, M0vsM6Forres1 = Treat1.6-Treat1.0, levels=design) fit2 <- contrasts.fit(fit, cm) fit3 <- eBayes(fit2)
I think the first question should be answered by topTable(fit3, coef="res1vsres0ForM0",adjust="fdr").
The third question should be answered by topTable(fit3, coef="M0vsM6Forres1",adjust="fdr") and topTable(fit3, coef="M0vsM6Forres0",adjust="fdr"), respectively.
However, I don't know how to answer the second question by LIMMA. I'd appreciate it if someone could help me. Thank you!