Additional file 1 of mRNA expression analysis of the hippocampus in a vervet monkey model of fetal alcohol spectrum disorder

Additional file 1: Supplemental Figure 1. RNA degradation plots showing 5’-3’ mean intensity levels for all 24 arrays revealing a similar slope across all samples without any major outliers as a result of 5’-3’ degradation. Supplemental Figure 2. Principal component analysis (PCA) of all 24 arrays for the unique expressed probe sets that remained after culling for annotation, multiple probe sets and MAS5 calls. Twenty-two arrays grouped together with FASD5_1 and FASD2_5 showing a distinct expression pattern which skews and distinguishes them from the other arrays due to non-technical variance. These arrays were excluded from further analyses to avoid skewing the group means due to variance unrelated to experimental factors. Supplemental Figure 3. 3D PCA plot of expression post normalization via RMA after exclusion of FASD5_1 and FASD2_5 revealing the unsupervised organization of the 22 remaining samples. Supplemental Figure 4. Box plots of GeneChip Rhesus Macaque genome array expression data for all 24 arrays after RMA normalization. Supplemental Figure 5. Histogram of p-value distributions for Alcohol divided into 20 bins with each bin representing 0.05 units. The distribution shows a distinct and sharp increase in the 0-0.05 bin indicating that Alcohol as an experimental factor resulted in a higher number of differentially expressed genes than would have been predicted under the null hypothesis. The black shaded bar represents the most frequent bin within the distribution. Supplemental Figure 6. Histogram of p-value distributions using Age as a main effect divided into 20 bins with each bin representing 0.05 units of distribution. The distribution shows a distinct and sharp increase in the 0-0.05 bin indicating that Age as an experimental factor resulted in a higher number of differentially expressed genes than would have been predicted under the null hypothesis. The black shaded bar represents the most frequent bin within the distribution. Supplemental Figure 7. Histogram of p-value distributions for the interaction between Age and Alcohol divided into 20 bins with each bin representing 0.05 units. The distribution shows a flat distribution indicating there is no evidence for a generalized genome wide interaction effect between these two experimental factors. The black shaded bar represents the most frequent within the distribution. Supplemental Figure 8. Correlation of log2 expression intensity and -delta Ct values using ACTB to normalize expression values. GBPB1L1 strays from the trend line implying that it may have amplified a target region not represented by the probe set. Supplemental Table 1. Mean intensity/expression levels (log2) and mean variance for all 11,512 mRNA for all four groups involved in this study. Supplemental Table 2. Primer sequences used for qRT-PCR amplification of selected genes taken from the Rhesus GeneChip array. Supplementary Table 3. Functional annotation results using the 297 genes which returned the lowest p-values using Age as a main effect. Eight of the 15 top annotation clusters returned results related to development or cell migration with lower overall p-values and FDR values when compared to those generated using alcohol as a main effect.

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