Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of human HUVEC IL-1 stimulated cells

ABSTRACT: 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 manufacturerâs protocols (https://www.affymetrix.com). Briefly, 5 Âµg 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 Âµg of cRNA was fragmented at 94Â°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 Âµl 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 Âµg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94Â°C for 5 minutes, equilibrated at 45Â°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 Âµg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45Â°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 25Â°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50Â°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.

ORGANISM(S): Homo sapiens

SUBMITTER: Herbert Mayer

PROVIDER: E-GEOD-973 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Deciphering regulatory patterns of inflammatory gene expression from interleukin-1-stimulated human endothelial cells.

Mayer Herbert H Bilban Martin M Kurtev Vladislav V Gruber Florian F Wagner Oswald O Binder Bernd R BR de Martin Rainer R

Arteriosclerosis, thrombosis, and vascular biology 20040506 7

<h4>Objective</h4>Endothelial cells comprise a key component of the inflammatory response. We set out to obtain a comprehensive overview of the immediate-early to early gene expression program of interleukin-1 (IL-1)-stimulated endothelial cells and to identify novel transcription factors and regulatory elements.<h4>Methods and results</h4>Human umbilical vein endothelial cells (HUVECs) were stimulated with IL-1 for 0, 0.5, 1, 2.5, and 6 hours and analyzed using Affymetrix U133 microarrays. A to ...[more]

PMID: 15130917

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Project 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 (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

Project description:Experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Microarray methodology follows: cDNA for each sample was synthesized from 8.5 Âµg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94Â°C for 35 minutes and chilled on ice. Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45Â°C with constant rotation (60 rpm). The arrays were washed and then stained for 10 min at 25Â°C with 10 Âµg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 Âµg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25Â°C. Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25Â°C. After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm. Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms. Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments. The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.

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Project description:We have used commercially available oligonucleotide arrays to measure the relative expression levels of >10,000 genes and ESTs. Recent evidence suggested certain advantages of performing such expression experiments on two or more different platforms of oligonucleotide arrays. Here we have used two oligonucleotide platforms, MG U74Av2.0 arrays from Affymetrix and CodeLink UniSet1 from GE Healthcare, to assess whole brain gene expression patterns in naive C57BL/6 and DBA/2J mice. C57BL/6J (n = 5) and DBA/2J (n = 4) male mice, 25-30 g body weight, were obtained from Jackson Laboratories. Animals were group-housed and quickly sacrificed by CO2 exposure according to a protocol approved by the UCHSC IACUC. Brains were rapidly removed and stored at -80oC until use. Whole brain was homogenized in lysis buffer using a Polytron, and total RNA was isolated using the RNeasy Midi Kit (Qiagen) following the protocol supplied by the manufacturer. An additional clean-up of total RNA was carried out using the RNeasy Mini Kit (Qiagen). Using the protocol supplied by the manufacturer, double-stranded cDNA was synthesized from the total RNA (4 ug of total RNA from an individual mouse brain was used for each array) and was used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was recovered using the RNeasy kit. Biotin-labeled cRNA from each mouse was then fragmented and hybridized with two separate (duplicate) CodeLink BioArrays. The average of the gene expression intensity values for each probe set from these technical replicates was used for data analysis. The BioArrays were subsequently stained for 30 min with Cy5-Streptavidin and washed prior to scanning. For MG U74Av2 arrays,double-stranded cDNA was synthesized from 5 ug of total RNA, using the protocol supplied by the manufacturer, and used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was then fragmented and the quality of the fragmented cRNA was assessed using Test2 chips prior to hybridization with the Affymetrix arrays. Hybridization of the cRNA with the Affymetrix arrays was carried out according to the manufacturer's protocol. The arrays were stained with streptavidin-phycoerythrin conjugate and scanned by a GeneArray scanner. Keywords: other

Project description:TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood. In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. 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The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 Âµg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 Â°C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs. Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 Â°C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 Â°C for 5 min, equilibrated at 45 Â°C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 Â°C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 Â°C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 Â°C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david).

Dataset Information

Transcription profiling of human HUVEC IL-1 stimulated cells

Publications

Deciphering regulatory patterns of inflammatory gene expression from interleukin-1-stimulated human endothelial cells.

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