The gravid uterus was removed and placed in a sterile PBS (0.1 M phosphate buffer containing saline) at 37°C. The embryo in the visceral yolk sac along with a small piece of the ectoplacental cone (hereafter called embryo, unless otherwise stated) was carefully removed from the deciduas tissues and the Reichert’s membrane and immediately immersed in PBS containing 4% fetal bovine serum (Sigma, St Louise. MO). Three embryos bearing 3-5 somites (E8.25) were placed in a culture bottle (20 mL, B.T.C. Precision Incubator Unit, B.T.C. engineering, Cambridge, England, 36 rpm) containing the culture medium which consisted of 70% immediately centrifuged heat-inactivated rat serum (Harlan Sprague-Dawley, Inc, Indianapolis, IN) and 30% phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 8 mM Na2HPO4, 1.47 mM KH2PO4, 0.9 mM CaCl2, 5.6 mM glucose, 0.33 mM sodium pyruvate, pH7.4) supplemented with 20 units/ml penicillin and 20 units/ml streptomycin (Sigma, St. Louis, MO). Bottles were gassed between 0 and 22 h with 5% O2, 5% CO2, and 90% N2, and between 22 and 44 h with 20% O2, 5% CO2, and 75% N2 in a rotating culture system (B.T.C. Precision Incubator Unit, B.T.C. engineering, Cambridge, England, 36 rpm) at 37 °C. After the pre-culture period (4 hours), alcohol exposure was started by transferring the embryos into medium containing 6 µL/mL of 95% ethanol (400 mg/dL in culture). The dose of alcohol exposure was previously determined decline from this initial peak concentration over the day to ~44mM (200mg/dL).
Extracted molecule
total RNA
Extraction protocol
Each embryo was immediately lysed in 1 ml RNeasy solution (TelTest, Houston, TX) and passed through a 18 gauge hypodermic needle 4 times. RNA was isolated according to the manufacturer’s directions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA (Expression Analysis Technical Manual, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 17 hr at 45C on RGU34A Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station.
Scan protocol
GeneChips were scanned using the standard Affymetrix protocol.
Description
Gene expression data from whole embryo
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1000.
