Hi, I have did an analysis like limma 8.8 section "Time Course Experiments" I will paste the text here and after I will make a question. > lev <- c("wt.0hr","wt.6hr","wt.24hr","mu.0hr","mu.6hr","mu.24hr") > f <- factor(targets$Target, levels=lev) > design <- model.matrix(~0+f) > colnames(design) <- lev > fit <- lmFit(eset, design) Which genes respond at either the 6 hour or 24 hour times in the wild-type? We can find these by extracting the contrasts between the wild-type times. > cont.wt <- makeContrasts( + "wt.6hr-wt.0hr", + "wt.24hr-wt.6hr", + levels=design) > fit2 <- contrasts.fit(fit, cont.wt) > fit2 <- eBayes(fit2) > topTableF(fit2, adjust="BH") Any two contrasts between the three times would give the same result. The same gene list would be obtained had "wt.24hr-wt.0hr" been used in place of "wt.24hr-wt.6hr" for example. Which genes respond in the mutant? > cont.mu <- makeContrasts( + "mu.6hr-mu.0hr", + "mu.24hr-wt.6hr", + levels=design) > fit2 <- contrasts.fit(fit, cont.mu) > fit2 <- eBayes(fit2) > topTableF(fit2, adjust="BH") Which genes respond differently in the mutant relative to the wild- type? > cont.dif <- makeContrasts( + Dif6hr =(mu.6hr-mu.0hr)-(wt.6hr-wt.0hr), + Dif24hr=(mu.24hr-mu.6hr)-(wt.24hr-wt.6hr), + levels=design) > fit2 <- contrasts.fit(fit, cont.dif) > fit2 <- eBayes(fit2) > topTableF(fit2, adjust="BH") topTableF give me something like this: ID wt.0hr wt.6hr F P.Value adj.P.Val 248 B-1:7 -0.27 -3.23 27.30 4.16e-08 0.000 360 B-2:9 -0.08 -2.62 23.10 2.56e-07 0.000 1999 I-16:8 -0.82 3.00 19.11 1.72e-06 0.001 487 C-1:6 -0.02 -2.92 18.99 1.82e-06 0.001 201 A-6:1 1.21 -3.36 18.92 1.89e-06 0.001 What the values in the columns wt.0hr and wt.6hr means? I could consider these values like fold change? For example, gene B-1:7 is 3.23 log fold change down regulate in time 6hr in relation to time 0hr. And in relation to time 24hr? If the contrasts "wt.24hr-wt.0hr" and "wt.24hr-wt.6hr" are equals, how I could know if gene B-1:7 is more expressed in time 6 than time 24, for example? Is there a way to plot a graph with expression in y-axis and time on x-axis for differentially expressed genes? How I could do this for this example? Thank you very much -- Marcelo Luiz de Laia Universidade do Estado de Santa Catarina UDESC - www.cav.udesc.br Lages - SC - Brazil Linux user number 487797 Please avoid sending me Word or PowerPoint attachments. See: http://www.gnu.org/philosophy/no-word-attachments.html http://www.gnu.org/philosophy/no-word-attachments.pt-br.html "Qual ? a minha expectativa, e por que eu sou petista, e por que com todos os desastres deste partido, eu continuo nele? Porque acho que temos um processo hist?rico lento a realizar, que come?ou muito antes de mim, e que os meus bisnetos v?o finalizar." Marilena Chaui