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Endothelial cells and atorvastatin

ABSTRACT: Rationale: Immortalized cells may exhibit important differences relative to their primary cell counterparts. Microarrays were used compare primary human umbilical vein endothelial cells (HUVECs) and the immortalized HUVEC cell line EA.hy926, in their response to inhibition of the mevalonate pathway by a HMG-CoA reductase inhibitor (atorvastatin). The effects of atorvastatin were reversed by the addition of mevalonate, to subtract non-specific changes in gene expression. Methods: Confluent cell cultures of HUVECs and EA.hy926 cells (two independent experiments in each cell type) were incubated with 1) statin-free media; 2) 10 microM atorvastatin; 3) 500 microM mevalonate; or 4) a combination of 10 microM atorvastatin and 500 microM mevalonate. All cells were harvested at 24 h. Total RNA was isolated using Trizol reagent (Invitrogen). cDNA was prepared from 10 microgram total RNA using a double-stranded cDNA synthesis kit (Life Technologies) with a T7-dT24 primer for first-strand synthesis. cRNA was synthesized from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). Twenty microgram of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer, containing 40 mM Tris-acetate, pH 8.1, 125 mM KOAc, 30 mM MgOAc. Fifteen micrgram of fragmented cRNA, together with control cRNAs and grid alignment oligonucleotides, were hybridized overnight to GeneChip Human Genome U133A 2.0 arrays (Affymetrix) at 45°C under constant rotation. Arrays were washed and incubated with an anti-streptavidin antibody. Fluorescent signals were measured using an Agilent Gene Array Laser Scanner and analyzed with MicroArray Suite 5.0 (Affymetrix). Microarrays were scaled using default settings. Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE2450 | GEO | 2005/03/29

SECONDARY ACCESSION(S): PRJNA92661

REPOSITORIES: GEO

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Project description:Experiment design Type of experiment: Gene expression profiling analysis of S.cerevisiae W303-1a. Experimental factor: genetic variation (WT, gpr1 and gpa2 mutant strains). Number of hybridizations: 6. Single-colour labeling was performed for each sample. Sample preparation: Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturers' instructions. Labeling protocol 10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Hybridization protocol 10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was controled by use of the GeneChip™ Eukaryotic Hybridization Control Kit (Affymetrix). Washing and staining was performed in a Fluidics Station 400 (Affymetrix) using the protocol EukGE-WS2v4. Measurement data, specifications and data processing Scanning was performed in an Affymetrix GeneChip scanner. Chip analysis was performed using the Affymetrix Microarray Suite v5. Changes in gene expression were assessed by looking for concordant changes between all replicates using a signed Wilcoxon rank test. The “change” p-value threshold was < 0.025 for increase and > 0.975 for decrease. Array design Microarray analysis was performed using YGS98 GeneChips™ oligonucleotide arrays (Affymetrix #900256) representing approximately 6000 ORFs. Sequences and GenBank accession numbers of all probe sets are available at the Affymetrix home page http://www.affymetrix.com/index.affx. Keywords: ordered

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 µg 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 94°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 45°C with constant rotation (60 rpm). The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C. Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°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. Keywords: other

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Endothelial cells and atorvastatin

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