After Experiment 2, we decided to test the three groups (control, ALC/NTO, ALC/NTC) as pools, and chose growth/neurotrophic genes. A separate experiment was carried out with embryonic treatments identical 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 different 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 Bulletin #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 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.
