Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of human neuroblastoma tumor-initiating cells treated with compounds identified in stem cell-based small molecule screens

ABSTRACT: Neuroblastoma Tumor-Initiating Cells (NB12, NB88R2, NB122) were treated with 100nM dequalinium, C-14 analog (DECA-14) or DMSO for 24 hours. Cells were collected, lysed in Trizol and RNA was purified with Rneasy mini kit. Single-stranded cDNA was generated from the amplified cRNA with the WT cDNA Synthesis Kit (Affymetrix) and then fragmented and labeled with the WT Terminal Labeling Kit (Affymetrix). Samples were hybridized with Affymetrix GeneChip Human Gene 1.0 ST Arrays and scanned at the HSC Microarray Facility. Array scanning was performed according to the manufacturer's instruction using Affymetrix GeneChip Scanner 3000. The data were background corrected and normalized using standard RMA procedure implemented in the Affymetrix Expression Console software. The preprocessed data were analysed using the LIMMA Bioconductor package to identify genes that show significant evidence of differential expression

ORGANISM(S): Homo sapiens

SUBMITTER: Kristen Smith

PROVIDER: E-MEXP-2835 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Transcription profiling by array of human neuroblastoma tumor-initiating cells treated with compounds identified in stem cell-based small molecule screens

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