Transcriptional analysis of WT and RasGrf1 KO pancreatic islets by microarrays. (A) Quality analysis of islet RNA used in the microarrays. Real-time PCR analysis comparing Ct values of equal cDNA amounts from islet or total pancreas RNA. Bars represent the islet/total pancreas ratios of signals produced by specific oligonucleotides for amylase (grey bar), insulin 1 (black bar) or insulin 2 (clear bar), normalized to beta-2-microglobulin (unchanged in all conditions). Ratios are presented in a log scale. (B) Graphical display of statistical analysis performed in islet RNA from RasGrf1 KO microarray data compared to WT. The plot compares observed values against expected values in SAM-contrasting analyses of six independent microarray hybridizations. Distance of a spot from the no-change diagonal means differential expression for a probeset and it is measured by the d-value. Quantitative cutoff values and FDR parameters defined by the horizontal and diagonal dashed lines are specified in the upper left corner. (C) Hierarchical clustering of the 4525 probesets showing differential expression in RasGrf1 KO islets (listed in Table S1, FDR 0.084; threshold p-value: 0.046). Dendrogram was generated by cluster analysis of absolute expression values of probesets. Each row represents single probeset expression. Columns identify results from single microarray hybridizations. Each box represents the hybridization signal value of a probeset in the corresponding microarray assay. The intensity of color in each box provides a quantitative estimation of its expression change. Blue: repressed probesets. Red: overexpressed probesets. Black: unchanged expression. Most significant KEGG pathways and associated p-values for horizontal clusters: Clusters 1a and 1b: MAPK signaling (7.92E-4 and 2.3E-3 respectively) and Calcium signaling (1.9E-3 and 2.7E-3, respectively). Cluster 1c: cytokine-cytokine interaction genes (p = 7.2E-4). Cluster 3: metabolic processes, including riboflavin metabolism and Kreb’s cycle (Cluster 3a, p = 6.7E-4 and 1.6E-3) or pyruvate metabolism (Cluster 3b, p = 7E-3).