Hi, While finding differential gene expression of Affymetrix data, I am unable to fix a small problem. The non-differentially regulated data are showing bigger in the subsequent MA-plot. Please correct me. Thanks, Santana = = = = = = = == = myRMA <- justRMA() number_genes <- dim(exprs(myRMA))[1] ### Filtering out uninteresting data is_control <- logical(number_genes) for (row in 1:number_genes) { if (charmatch("AFFX", rownames (exprs(myRMA))[row], nomatch=0) == 0) is_control[row] <- TRUE else is_control[row] <- FALSE } myRMA_no_controls <- myRMA[is_control] # Filtering out the least variable genes, defined by the 90th percentile of the distribution of CV-values sd_values <- apply (exprs(myRMA_no_controls), MARGIN=1, FUN="sd") mean_values <- apply (exprs(myRMA_no_controls), MARGIN=1, FUN="mean") CV_values <- sd_values/mean_values quantile_cut_off <- quantile(CV_values, probs=0.9) myRMA_filtered <- myRMA_no_controls[CV_values>quantile_cut_off] design <- cbind (Healthy = c(0,0,0,0,0,0,0,1,1,1,1,1,1,1,1,1,1), Diseased=c(1,1,1,1,1,1,1,0,0,0,0,0,0,0,0,0,0)) fit <- lmFit (myRMA_filtered, design) contrasts_matrix <- makeContrasts(Healthy-Diseased, levels = design) fit2 <- contrasts.fit (fit, contrasts_matrix) fit3 <- eBayes (fit2) number_genes <- dim(exprs(myRMA_filtered))[1] test_results <- topTable(fit3, number=number_genes, adjust="BH") # False discovery rate cut off set to 0.05. FDR_cutoff <- 0.05 p_values <- fit3$p.value adjusted_p_values <- test_results$adj.P.Val # Identify significant genes : significant_genes <- test_results[test_results$adj.P.Val <= FDR_cutoff,] gene_index <- rownames(significant_genes) # MA-plot displaying the log fold change between diseased and healthy samples as a function of the average expression level across all samples. status <- character (length=number_genes) status <- rep ("not changing", number_genes) names (status) <- seq (1,number_genes,1) status [gene_index] <- "significant" plotMA (fit3, status=status, col=c("blue","pink")) # the pink data- ponits are coming relatively bigger > sessionInfo() R version 2.10.1 (2009-12-14) i386-pc-mingw32 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] hgu133acdf_2.5.0 limma_3.2.1 affycoretools_1.18.0 annaffy_1.18.0 KEGG.db_2.3.5 GO.db_2.3.5 RSQLite_0.8-4 [8] DBI_0.2-5 AnnotationDbi_1.8.0 affy_1.24.1 Biobase_2.6.0 loaded via a namespace (and not attached): [1] affyio_1.14.0 annotate_1.24.0 biomaRt_2.2.0 Biostrings_2.14.2 Category_2.12.0 gcrma_2.18.0 genefilter_1.28.0 [8] GOstats_2.12.0 graph_1.24.1 GSEABase_1.8.0 IRanges_1.4.3 preprocessCore_1.8.0 RBGL_1.22.0 RCurl_1.3-1 [15] splines_2.10.1 survival_2.35-8 tools_2.10.1 XML_2.8-1 xtable_1.5-6 [[alternative HTML version deleted]]