Gene expression Profiling of Peripheral Blood Leukocytes after Endotoxin Challenge in Humans

Peripheral blood mononuclear cells were isolated 0, 30 min, 6 h, 24 h, and 7 day after intravenous endotoxin challenge. Keywords: time course Peripheral blood mononuclear cells were isolated 0, 30 min, 6 h, 24 h, and 7 day after intravenous endotoxin challenge using cell preparation tubes (Vacutainer® CPT). The PBMC layer was lysed with RLT buffer, homogenized, and then stored at –80°C. Total RNA was extracted as per RNeasy‚ Midi protocol (Qiagen). Double stranded cDNA was synthesized from total RNA (5 to 20 µg) using 7-d(T)24 primer and SuperScript‰ Double-Stranded cDNA Synthesis Kit and purified by phase lock gel-phenol/chloroform extraction followed by ethanol precipitation. Biotin-labeled cRNA was prepared by in vitro transcription using HighYield‰ RNA Transcript Labeling Kit followed by fragmentation with 5X fragmentation buffer. Fragmented cRNA (10 µg) was hybridized to Affymetrix Hu95Av2 oligonucleotide probe arrays for 16 h at 45∞C. After removal of hybridization fluid, the arrays were washed, stained with streptavidin phycoerythrin (SAPE), and signal amplified by anti-streptavidin antibody using Affymetrix Fluidics Station 400. Probe set signals were measured using Agilent GeneArray Scanner (Affymetrix‚, Santa Clara, CA).