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Proteins were transferred to ImmobilonFL PVDF membranes (Millipore, Billerica, MA) and probed with an anti p53 mouse monoclonal antibody at 1:1000 (clone 1C12, #2524, Cell Signaling Beverly, MA). The immunoblot was imaged with HRP conjugated anti mouse IgG secondary at 1:2500 (Promega, [http://www.selleckchem.com/products/cpi-1205.html CPI-1205 mouse] Madison, WI) and peroxide/luminol chemiluminescence with the SuperSignal West Pico kit (Pierce/Thermofisher, Rockford, IL). For loading control blots were re-probed with mouse monoclonal anti-GAPDH at 1:5000 (AM4300, Ambion, Foster City, CA) and imaged with goat anti mouse secondary conjugated to IR800 fluorophore at 1:5000 on the LICOR Odyssey system (Lincoln, NE). Three samples per phenotypic class were analyzed. Student's t-tests and Pearson's correlations of Q-PCR data were performed in Prism 4 (GraphPad Software, LaJolla, CA). Significance was set at p?[http://en.wikipedia.org/wiki/DEF6 DEF6] severe midline fusion ( Fig.?1A). To determine if these phenotypic classes are associated with differences between their transcriptomes, we used mouse exon microarrays to analyze total RNAs isolated from BA1 samples at E10.5. Differences in gene expression between the classes increased with increasing phenotypic severity, and thus were greatest in the most severely affected (class C) embryos ( Fig.?1B). Hierarchical clustering analysis was performed on the transcript expression profiles of a set of 127 genes exhibiting the greatest differences in expression between any two pools (Fig.?1C). This analysis clearly distinguished class C phenotypic samples from WT and class A samples. In fact, the clustering algorithm placed class A embryos just as distantly from WT as class C from WT, implying that gene expression differences in class A embryos, albeit not as pronounced as in [http://www.selleckchem.com/products/mk-4827-niraparib-tosylate.html MK-4827 solubility dmso] class C, may be sufficient to compensate for the Twsg1?/? genetic predisposition. Class B samples, representing an intermediate phenotype, clustered alternatively with either class A or class C. These results demonstrate that the variable morphological features of individual embryos with the same genetic mutations in Twsg1 on the same genetic background are associated strongly with distinct transcriptional profiles. To study the association between phenotypic variability and differential gene expression in more detail, we first focused upon samples representing the most divergent morphological classes, i.e. WT vs. class C embryos.