E-GEOD-3026 - Transcription profiling of human peripheral blood mononuclear cells, or whole blood were isolated 0, 30 min, 6 h, 24 h, and 7 day after intravenous endotoxin challenge vs controls
Submitted on 28 July 2005, released on 19 December 2007, last updated on 27 March 2012
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).
transcription profiling by array, cell type comparison, co-expression, compound treatment, individual comparison, sex, time series
Gene expression profiles of peripheral blood leukocytes after endotoxin challenge in humans. Shefali Talwar, Peter J Munson, Jennifer Barb, Carmen Fiuza, Anadel Pilar Cintron, Carolea Logun, Margaret Tropea, Sameena Khan, Debra Reda, James H Shelhamer, Robert L Danner, Anthony F Suffredini. Physiol Genomics 25(2):203-15 (2006)