PolyA RNA was purified twice from cytoplasmic RNA (for quiescent, regenerating and newborn livers, and liver tumor samples) using the Oligotex mRNA Isolation Kit (Qiagen). The polyA RNA (1 µg) was used to create cDNA with a T7-polyT primer and reverse transcriptase Superscript II (GIBCO/BRL). Approximately 1 µg of cDNA was subjected to in vitro transcription (Ambion) in the presence of biotinylated UTP and CTP (Enzo Diagnostics) for 4 hours at 37°C. The IVT reaction products was cleaned using the RNeasy Mini Kit (Qiagen). The cRNA was fragmented at 94°C for 35 min in 5X RNA Fragmentation Buffer [200 mM Tris-acetate, 500 mM KOAc, 150 mM MgOAc]. The fragmented cRNA is referred to as the target. The target was prepared for hybridization by combining 40 µg of fragmented cRNA with sonicated herring sperm DNA (0.1 mg/ml), BSA (0.5 mg/ml), and a buffer containing 2X MES, 1.7 M NaCl, 40 mM EDTA, and .02% Tween 20. The 100X control cRNA (BioB, BioC, BioD, cre) and control Oligonucleotide B2 (5 nM) were also added to the target hybridization mix. Target was hybridized for 16 hr at 40°C to each oligonucleotide array (MU6500 tetraset; Affymetrix) containing probes for 6500 genes and ESTs. Arrays were washed at 50°C with 6X SSPE-T [0.9M NaCl, 60 mM Na2HPO4, 6 mM EDTA, 0.005% Triton X-100, pH 7.6] then at 40°C with 0.5X SSPE-T. Arrays were then stained with streptavidin phycoerythrin (Molecular Probes). Arrays were washed with 6X SSPE-T in a fluidics station and fluroescent intensities were measured with a laser confocal scanner (Hewlett-Packard) and analyzed with Gene Chip Expression Algorithm (Affymetrix).
· Complete details can be found in the Affymetrix Protocols.
· These experiments were performed at Pfizer Global (formerly known as Parke-Davis) with the guidance of Jim Messamore, Erika Perguson, Tim Jatkoe, and Dr. Steve Madore.