Hierarchical clustering with average linkage function was used to construct a dendrogram based upon all genes that were present on at least half of the arrays in an experimental group. Gene Set Enrichment Analysis was carried out to identify groups of related genes Rapamycin purchase that were differentially expressed. GSEA analyses were conducted for 4 different comparisons, control vs. ALC, control vs. ALC NTC, control vs. ALC NTO, and ALC NTC vs. ALC NTO. The top ranked genes in a significant gene set, in the region up to the maximum score, were con sidered significant. To reduce multiple testing issues, the GSEA in this study was conducted using two gene set databases designed to test the hypotheses that groups of genes related to Early Development or Stem Cells were differentially affected by alcohol.

Early Developmental Biology Gene Sets, 415 GO categories that were defined by 29 key words were selected. Stem Cell Related Gene Sets, 191 GO categories related to stem cells, neurogenesis, osteogenesis, extra cellular matrix, developmental signal transduction path way, cell cycle, growth factor, TGFb BMP signaling, Wnt signaling, and notch signaling were developed by Superarray Bioscience. The gene set information is listed in Additional file 3. Quantitative Real Time Polymerase Chain Reaction A number of differentially expressed genes detected in Experiment 1 were selected for qRT PCR validation based on their biological significance. To test selected genes from the neural specification gene group, the total RNA of each embryo was isolated using the RNeasy mini kit as described above.

Vec tor NTI Advance 9. 0 software was used to design the primers for qRT PCR, if possible, at least one primer in each pair spanned an exon intron boundary. The number of embryos used in the control group varied from 7 to 9 for different genes, and the number used in the alcohol treated group varied from 9 to 11. The cDNA templates were generated from 50 ng total RNA from each individual embryo, and added to PCR reactions that contained 0. 1 uM of forward and reverse primers and SYBR Green PCR Master Mix. Triplicate qRT PCR were performed for each sample in at least 3 experiments. The cycle threshold for each cDNA template was determined on the ABI Prism 7700 Sequence Detection System. The Ct refers to the cycle number at which the fluorescence of the amplified product reached an arbitrary threshold that was within the exponential phase of amplification. Entinostat To correct for sample to sample variation, Gapdh served as an internal reference. Relative values of expression of neural specific genes were determined for each sample using the Ct method, and these values were normalized to the Ct values of Gapdh.