Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 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 (Qiagen, Valencia, CA) as described above. Vec- tor NTI Advance 9.0 software (Invitrogen, Frederick, MD) was used to design the primers for qRT-PCR (Table 8); 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 (TaqMan Reverse Transcription Reagents, Applied Biosystems, Foster City, CA) from each individual embryo, and added to PCR reactions that contained 0.1 μM of forward and reverse primers and SYBR Green PCR Master Mix (Applied Biosystems). Triplicate qRT-PCR were performed for each sample in at least 3 experiments (n = 9). The cycle threshold (Ct) 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. 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 ΔΔCt method [76], and these values were normalized to the Ct values of Gapdh. The average Gapdh Ct values for alcohol treatment and con- trol were the same in each tested sample, making it an appropriate control gene to normalize the expression of the candidate genes of interest. After Experiment 2, we decided to test the three groups (control, ALC/NTO, ALC/NTC) as pools, and chose growth/neurotrophic genes. A separate experi- ment was carried out with embryonic treatments identi- cal to those used in Experiment 1. Whole embryos were homogenized in TRIzol (Invitrogen) using a Mini-Bead- Beater-8 (Bipspec products, INC, Bartlesville, OK), and total RNA isolation was as described above. Two differ- ent pools were created for each condition: Control1 (n = 12), ALC/NTC1 (n = 16), ALC/NTO1 (n = 5), Control2 (n = 5), ALC/NTC2 (n = 9), ALC/NTO2 (n = 6). The relative quantification of expression of each RNA pool was performed using the ABI Prism 7700 Sequence Detection System and calculated using the standard curve method (Applied Biosystems, User Bulle- tin #2; http:////www.appliedbiosystems.com). In each experiment, a relative expression level was determined for the two pools from each group in triplicate; 3-4 Page 15 of 17 repeat experiments were performed, resulting in 18-24 values from each group. The treatment groups were compared with one way ANOVA followed by Student’s t test. Additional material Additional file 1: Keyword and GO categories. List of keywords used and the GO categories identified by these key words for Early Development. Additional file 2: GO gene sets selected by keywords. List of gene sets used in GSEA analyses, derived from GO categories selected by key words for Early Development. Title of each set followed by Entrez Gene Ids of the genes in the set. Additional file 3: GO categories related to Stem Cells. Gene sets used in GSEA analysis based on GO categories related to Stem Cells. Title of each set followed by Entrez Gene Ids of the genes in the set. Acknowledgements This study was supported by NIAAA P50 AA07611 to the Alcohol Research Center at Indiana University School of Medicine (Center PI, David W. Crabb; FAS project, Feng C. Zhou), and in part by AA016698 to FCZ. Microarray studies were carried out in the Center for Medical Genomics at Indiana University School of Medicine, which is partially supported by the Indiana Genomic Initiative at Indiana University (INGEN®); INGEN® is supported in part by the Lilly Endowment, Inc. We thank Ms. Li-jun Ni for her technical assistance in embryonic culture, and Ronald Jerome and Chunxiao Zhu for technical assistance in preparation of microarray. Author details 1Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, 46202, USA. 2Department of Medicine, Indiana University School of Medicine, 545 Barnhill Drive, Indianapolis, IN, 46202, USA. 3Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA. 4Department of Psychology, Indiana University- Purdue University at Indianapolis 402 N. Blackford Street, Indianapolis, IN, 46202, USA. Authors’ contributions FCZ involved in the overall design and coordination of the study. CG involved in the design of the experiment. QZ and LL carried out the statistical analysis, and YL the informatics. TL performed RTPCR analysis. HJE and JNM were involved in experimental design and carried out the microarray study. All authors participated in the writing of the manuscript. All authors read and approved the manuscript. 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