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. Vector 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
