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NT2 DMSO vs. RA

ABSTRACT: NT2 cells were treated with DMSO (control) or 10uM Retinoic Acid (RA). Total RNA was isolated using TriReagent, and subsequent cleanup with RNeasy columns (Qiagen) according to the manufacturer's directions. Hybridizations were performed according to Affymetrix guidelines using the Human HG-U133A chip containing 22,500 unique genetic elements. Double-stranded cDNA was synthesized using Superscript II RT (Invitrogen). In vitro transcription labeling was performed with biotin labeled nucleotides using the Bioarray High Yield RNA Labeling Kit (ENZO). 20 ul of labeled RNA per sample was added to the hybridization cocktail, with a total of 15 ug hybridized to the chip (16 hours, 45C). RMA analysis was performed using Gene Traffic software (Iobion), where raw data for each hybridization was normalized, background corrected and filtered for signal intensity of 50 or greater in at least one comparison. Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE2448 | GEO | 2005/10/18

SECONDARY ACCESSION(S): PRJNA92657

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

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NT2 DMSO vs. RA

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