Total RNA was isolated from two batches of 6 to 10 LVCP, sampled from E19, P2, and adult rats, using the RNeasy® Micro Kit (Qiagen, Valencia, CA, USA), and was DNase-treated according to the manufacturer's protocol. Total RNA was quantified using OD 260nm on a NanoDrop 2000c spectrophotometer (ThermoScientific, Baltimore, MA, USA) and quality was assessed with the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). 100 ng of total RNA were amplified using the GeneChip 3' IVT Express Kit (Affymetrix).
Label
biotin
Label protocol
Biotinylated cRNA was prepared according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Hybridization was performed according to the Affymetrix protocol (http://www.affymetrix.com). Briefly, 10 µg of labelled cRNA was fragmented and denaturated in hybridization buffer, then hybridized on a chip during 16 hours at 45°C with constant mixing by rotation at 60 rpm in a GeneChip hybridization oven 640 (Affymetrix). After hybridization, arrays were washed and stained with streptavidin-phycoerythrin (GeneChip® Hybridization Wash and Stain Kit) in a Fluidic 450 (Affymetrix) according to the manufacturer's instructions.
Scan protocol
The arrays were read with a confocal laser (GeneChip scanner 3000, Affymetrix). Then, CEL files were generated using the Affymetrix GeneChip Command Console (AGCC) software 3.0.
Description
Sprague-Dawley rat_E19-2. Gene expression data from CP of embryos.
Data processing
The obtained data were normalized with Affymetrix Expression Console software using the MAS5 statistical algorithm. Two batches of animals were used for each stage and the mean value of both was used for further analysis.
