Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 20 units/ml streptomycin (Sigma, St. Louis, MO), and gassed with 5% O2, 5% CO2, and 90% N2 in a rotating culture system (B.T.C. Precision Incubator Unit, B.T.C. engineering, Cambridge, England, 36 rpm) for 2 h. After 2 h, treatment was initiated by transferring embryos into the same medium with or without 88 mM ethanol in isotonic buffer. The bottles were gassed for an addi- tional 20 h with 5% O2, 5% CO2, and 90% N2, and then between 22 h and 46 h with 20% O2, 5% CO2, and 75% N2. The culture medium in alcohol and control cultures was replaced with fresh medium (with or without etha- nol, respectively) 22 h after the start of the treatment. In this culture system, it was previously determined that the media alcohol concentration declined from 88 mM to 44 mM over the course of the experiment. Alcohol concentrations in this range (44-88 mM) have been commonly used in whole embryo cultures to generate FAS-related structural malformations [41,42,67] in mul- tiple strains of mice [29], and are comparable to blood alcohol concentrations produced by in vivo doses of acute ethanol injections that produce teratogenic effects in mice during this embryonic period [68]. This level, though high, is within the range attained by human alcoholics [69,70]. All cultures were terminated 46 hrs from the begin- ning of treatment. The concentration of ethanol in the medium was assayed at three time points on each day (0 [initial], 12, and 22 hours on the first day; at 0 [after media change], 12, and 24 hours on the second day) in a separate group of embryos not used for the analyses, to avoid the potential confounding effects of drawing samples from the cultures. Media samples from alcohol- or vehicle-treated cultures were assayed in duplicate for alcohol concentrations using an Analox alcohol analyzer (Analox Instruments USA, Lunenburg, MA). At the end of culture, viability was confirmed by observing the blood circulation of the yolk sac and the beating heart. Cultured embryos were quickly immersed in 0.7 ml TRIzol (Invitrogen, Carlsbad, CA) and homo- genized for extracting total RNA for the RT-PCR and microarray processes (see microarray section, below), or fixed in 4% paraformaldehyde in PBS for the evaluation of the developmental status. Whole embryos were used because the dysmorphology is observed throughout tissue derived from the three germ layers and in various developing organs (e.g., head fold, caudal neural tube, heart, lung bud, somites, and limbs). Also, dissection of the millimeter size embryos would unavoidably introduce another source of variabil- ity: whole embryos yield sufficient total RNA for single embryo analysis, whereas dissected tissues yield too little RNA and would require pooling or amplification for microarray analysis. Although this limits the resolution of genes contributing to different topographic changes, Page 13 of 17 we thought that obtaining a complete gene expression profile in parallel with this widespread alcohol-induced teratogenesis in the embryo would be informative. Embryonic dysmorphology The analysis of embryo dysmorphology was performed as described by van Maele-Fabry et al. [71] and in our previous report [29]. The morphological features of the developing embryo, including the allantois, flexion, heart, caudal neural tube, hind-brain, midbrain, fore- brain, otic system, optic system, branchial bars, maxil- lary process, mandibular process, forelimb, hindlimb, and somites, were examined and scored for any malfor- mations using the ordinal scales of our previous report [29]. Scores for each of the above features were typically not normally distributed, so they were analyzed statisti- cally by the non-parametric Mann-Whitney U test. The number of somites was normally distributed, so those data were analyzed by Student’s t-test, using StatView software (SAS Institute, Inc. Cary, NC). Gene expression analyses Two microarray experiments were performed. In Experi- ment 1, total RNA was isolated from individual whole embryos (4 vehicle control, 4 alcohol treated). Each embryo was immediately immersed in 700 ml TRIzol (Invitrogen) and homogenized using a Polytron. Extrac- tion followed the TRIzol protocol. Ethanol precipitated RNA was resuspended in DEPC water. RNA was cleaned up using RNeasy mini-kit (Qiagen, Valencia, CA) The quality of RNA was assessed by the Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Ger- many)and by spectrophotometry from 220 nm to 350 nm; concentration was determined from A260. Typical total RNA yields were 5-10 μg/embryo. Microarray ana- lysis was performed at the Center for Medical Genomics at the Indiana University School of Medicine. Labeling and hybridization to Affymetrix Mouse Genome 430A GeneChips® (Affymetrix, Santa Clara, CA) were carried out following the manufacturer’s suggested procedure. Fragmented biotinylated RNA from each embryo was separately hybridized to its own GeneChip for 17 hours at 42°C. The microarray analysis revealed striking differ- ences among the 4 alcohol treated samples, which segre- gated as two separate pairs rather than one set of four; subsequently, it was noted that one pair of embryos had an open neural tube (ALC-NTO) and the other pair had the neural tube closed (ALC-NTC). All 4 control embryos had closed neural tubes. Experiment 2 was designed to follow-up these initial results and provide an independent test of the gene expres- sion correlations with the two neural tube phenotypes. Total RNA was isolated from individual embryos (4 vehicle control, 7 alcohol treated: 4 ALC-NTO, 3 ALC-NTC).