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Sample GSM15390 Query DataSets for GSM15390
Status Public on Jul 20, 2004
Title HUVEC 0,5hr IL1
Sample type RNA
Source name Total RNA from IL-1 stimulated HUVEC (0,5hr)
Organism Homo sapiens
Extracted molecule total RNA
Description 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 ( 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. The given sample record represents the 0,5 hr time point of IL-1 stimulation.
Submission date Jan 16, 2004
Last update date Oct 28, 2005
Contact name Herbert Mayer
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
Platform ID GPL96
Series (1)
GSE973 IL-1 stimulation of HUVEC

Data table header descriptions
VALUE Signal from MAS5.0
ABS_CALL Detection Call from MAS5.0

Data table
AFFX-BioB-5_at 253.6 P
AFFX-BioB-M_at 457.8 P
AFFX-BioB-3_at 190.9 P
AFFX-BioC-5_at 624 P
AFFX-BioC-3_at 336.1 P
AFFX-BioDn-5_at 467.7 P
AFFX-BioDn-3_at 4590.5 P
AFFX-CreX-5_at 4985.7 P
AFFX-CreX-3_at 8915.6 P
AFFX-DapX-5_at 30.8 A
AFFX-DapX-M_at 43.5 A
AFFX-DapX-3_at 15.7 A
AFFX-LysX-5_at 29.2 A
AFFX-LysX-M_at 13.7 A
AFFX-LysX-3_at 21.4 A
AFFX-PheX-5_at 4.2 A
AFFX-PheX-M_at 4.1 A
AFFX-PheX-3_at 14.4 A
AFFX-ThrX-5_at 10.2 A
AFFX-ThrX-M_at 29.9 A

Total number of rows: 22283

Table truncated, full table size 401 Kbytes.

Supplementary file Size Download File type/resource
GSM15390.CEL.gz 3.5 Mb (ftp)(http) CEL

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