Total RNA was extracted from the entire brain of NF-I-A-deficient and wild-type control mice (n=3 each) using TRIzol (Invitrogen) according to the manufacturer’s instructions. Further purification of RNA was performed with the RNeasy Mini Kit (Qiagen). Total RNA concentration was determined using a spectrophotometer at 260 nm and 280 nm wavelength. To check RNA integrity, total RNA was separated in a 1% agarose gel containing formaldehyde, and the intensity ratio of 28S and 18S ribosomal RNA bands was assessed after ethidium bromide staining.
Label
biotin
Hybridization protocol
Procedures for cDNA synthesis, labeling and hybridization were carried out according to the manufacturer's protocol (Affymetrix). All experiments were performed using Affymetrix mouse genome Genechip U74A version 2. Briefly, 15mg of total RNA were used for first strand cDNA synthesis with an HPLC-purified T7-(dT)24 primer. Synthesis of biotin-labeled cRNA was carried out using the ENZO RNA transcript labeling kit (Affymetrix). For hybridization, 15 µg of fragmented cRNA were incubated with the chip in 200 µl of hybridization solution in Hybridization Oven 640 (Affymetrix) at 45oC for 16 hours. Genechips were washed and stained with streptavidin-phycoerythrin using the microfluidic workstation™ (Affymetrix) and scanned with a laser scanner (Agilent Technologies).
