Human gene 1.0st genechips

Microarray services were provided by the UPenn Microarray Facility, including quality control tests of the total RNA samples by Agilent Bioanalyzer and Nanodrop spectrophotometry. All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 100ng of total RNA was converted into first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted into cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end-labeling. cRNA yields ranged from 36-89μg, and cDNA was added to Affymetrix hybridization cocktails, heated at 99°C for 5 min and hybridized for 16 h at 45°C to Human Gene 1.0ST GeneChips (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6× SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. A confocal scanner was used to collect fluorescence signal after excitation at 570 nm.