Total RNA was isolated with the guanidinium thiocyanate method, extracted with acid phenol and precipitated from 2-propanol.
Label
biotin
Label protocol
Using a poly-dT primer incorporating a T7 promoter, double-stranded cDNA was synthesized from 10 micrograms total RNA using a cDNA synthesis kit (SuperScript double-stranded cDNA synthesis kit; Invitrogen, Carlsbad, CA). Double-stranded cDNA was purified by phenol/chloroform extraction using a Phase-Lock Gel (PLG Heavy Brinkmann Instruments, Westbury, NY) followed by ethanol precipitation. Biotin-labeled cRNA was generated from the double-stranded cDNA template though in-vitro transcription with T7 polymerase, and a nucleotide mix containing biotinylated CTP and UTP (Enzo RNA Transcript Labeling Kit; Enzo Diagnostics, Farmingdale, NY). The biotinylated cRNA was purified using RNeasy affinity columns (Qiagen, Valencia, MD).
Hybridization protocol
For each GeneChip, 20 micrograms of the labeled product was fragmented in 40 mM Tris-acetate, pH 8.1, 100mM KOAc, 30mM MgOAc, for 35 minutes at 94 degrees-Celsius, to an average size of 35 to 200 bases. 15 micrograms of this fragmented, biotinylated cRNA, along with hybridization controls supplied by the manufacturer (Affymetrix), were hybridized to the arrays for 16 hours at 45 degrees-Celsius and 60 rpm. Arrays were washed and stained according to the standard Antibody Amplification for Eukaryotic Targets protocol (Affymetrix).
Scan protocol
The stained GeneChip arrays were scanned at 488 nm using an Affymetrix Gene Chip Scanner 3000 (Affymetrix, Santa Clara, CA).
Description
flow-cytometry: ND; clinical-signs: failure to nest
