E12.5 wild-type and Pax6 homozygous rat embryos were dissected from the uterus obtained from pregnant female rats under deep anesthesia. To prepare RNA of distinct brain regions, the rostral part of wild-type and Pax6 homozygous rat embryos were first cut off at the upper forelimb level, and then subdivided into two parts including the forebrain (FB) and the hindbrain (HB), respectively, by cutting at the isthmus. Thus, the FB tissue also included the eye and nose primordia and the midbrain, and the HB tissue included pharyngeal arches as well. Total RNA was prepared using TRIzol Reagent (Invitrogen) according to the manufacture’s instructions. mRNA was purified from 250µg of total RNA by OligotexTM-dT30 <Super> mRNA Purification Kit (Takara). Approximately 2µg of mRNA was reverse-transcribed by Superscript II reverse transcriptase (Invitrogen) with T7- (dT)24 primer containing a T7 RNA polymerase promoter sequence. Then, double strand DNA (dsDNA) was synthesized by SuperScriptTMChoice System (Invitrogen). 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 were synthesized by in vitro transcription reaction with BioArrayTMHighYieldTMRNA Transcript Labeling Kit (ENZO Diagnostics). 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 15 ug of target cRNA, control oligo BS (Affymetrix), and Eukaryotic Hybridization controls (Affymetrix)] was hybridized with a rat U34A array at 45 °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 on HP GeneArrayTM scanner. Experiments were repeated twice with independent mRNA preparation. To quantify the expression level of a transcript, the average difference value (Adv) was calculated from the intensities against the probe set using the global methods of normalization by Microarray Suite ver. 4.0 (Affymetrix). Keywords = Pax6 transcription factor Keywords = wild-type rat embryos Keywords = Pax6 homozygous mutant rat embryos Keywords = forebrain Keywords = hindbrain Keywords = up-regulated genes Keywords = down-regulated genes.
