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Series GSE973 Query DataSets for GSE973
Status Public on Jul 20, 2004
Title IL-1 stimulation of HUVEC
Organism Homo sapiens
Experiment type Expression profiling by array
Summary Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturer’s protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4) at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500.
Keywords: time-course
 
 
Contributor(s) Mayer H, Bilban M, Kurtev V, Gruber F, Wagner O, Binder BR, de Martin R
Citation(s) 15130917
Submission date Jan 16, 2004
Last update date Aug 10, 2018
Contact name Herbert Mayer
E-mail herbert.mayer@univie.ac.at
Phone +43 1 4277 62552
Fax +43 1 4277 62550
Organization name Institute of Vascular Biology and Thrombosis Research
Street address Brunnerstr 59
City Vienna
ZIP/Postal code A-1235
Country Austria
 
Platforms (1)
GPL96 [HG-U133A] Affymetrix Human Genome U133A Array
Samples (5)
GSM15389 HUVEC 0hr Ctl
GSM15390 HUVEC 0,5hr IL1
GSM15391 HUVEC 1hr IL1
Relations
BioProject PRJNA88013

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE973_RAW.tar 17.6 Mb (http)(custom) TAR (of CEL)
Raw data provided as supplementary file

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