Dissection of E16.5 cortex for RNA extraction. Cortex Hemispheres of E16.5 mice were divided into the medial (R1), dorsal (R2), ventral (R3), lateral (R4), and occipital regions (R5). Preparation of RNA and Synthesis of cDNA. Poly A+ RNA was obtained through twice column procedures with oligo dT cellulose column (New England Biolabs, Beverly, MA) and was used for synthesis of cDNA. 1ug of polyA+ RNA was converted into double-stranded cDNA by reverse transcription using ReverTra Ace (ToYoBo, Japan) with oligo dT(24) primers containing the T7 RNA polymerase promotor sequence (Amersham Pharmacia Biotech, Japan). The integrity of RNA was confirmed by agarose electrophoresis and RNA contents were analyzed by measuring the optical density at 260 nm. Target preparation and array processing. The following procedures including preparation of cRNA targets, hybridization, washing, staining, and scanning were performed as described in the Expression Analysis Technical Manual (Affymetrix, CA). Briefly, biotin-labeled antisense cRNA targets were synthesized in in vitro transcription reaction system (IVT) using cDNA as template (ENZO BioArrayTM HighYieldTM RNA Transcription Labeling kit with T7 RNA polymerase, Enzo, NY). The cRNA targets were size-fragmented to 35-200 bp in a buffer containing potassium and magnesium acetate at 94°C for 35 minutes and were used for hybridization as cRNA targets. The hybridization cocktail [containing 10 ug of target cRNA, control oligo BS (Affymetrix), and Eukaryotic Hybridization controls (Affymetrix)] was hybridized with a mouse U74A array at 42°C for 16 hours in GeneChip Hybridization Oven 640. Washing and staining were performed after hybridization under the fluidics station protocol, EuGE-WS2, on GeneChip Fluidics Station 400. Then, we scanned the array twice on HP GeneArrayTM scanner. Experiments were repeated twice with independent dissection. Data analysis. To quantify the expression level of a transcript, the average difference value (Adv) was calculated from the intensities against the probe cells using the global methods of normalization by Microarray Suite ver. 4.0 (Affymetrix).
