Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human colon cells induced to differentiation

ABSTRACT: cells are induced to differentiation by removal of growth factors and the addition of 10% fetal calf serum in the culture medium

ORGANISM(S): Homo sapiens

SUBMITTER: alfredo pagliuca

PROVIDER: E-MEXP-2562 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mouse ErythroLeukaemic (MEL) cell lines were maintained in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin. MEL cells in the log phase of growth (0.5 to 1 millions cells / mL) were induced to differentiate with 2% dimethylsulfoxide (DMSO) for four days. Total RNA was isolated in triplicate from non-induced and DMSO-induced MEL cells using RNeasy Mini Kit (Qiagen). Purity and quality of isolated RNA were assessed by RNA 6000 Nano assay on a 2100 Bioanalyzer (Agilent Technologies, Santa 6 Clara, CA). All samples showed a RIN>8 and were all used for subsequent labeling. RNA (300 ng) was used for production of end-labelled biotinylated ssDNA. Labelled ssDNA was hybridized to the GeneChip Mouse Gene 1.0 ST array oligonucleotide microarray (Affymetrix, Santa Clara, CA) according to manufacturers recommendations. To examine the quality of the various arrays, measured intensity values were analyzed using Gene Expression Console (Affymetrix, Santa Clara, CA). The analyses indicated a high quality of all samples and overall comparability. The microarray data was normalized by quantile normalization, and followed by a summarization using Tukeys median polish algorithm (Irizarry et al., 2003) in Expression Console from Affymetrix (Santa Clara, CA). Data was further processed using packages within the Bioconductor project in the R statistical programming environment (Gentleman et al., 2004). In order to normalize between experimental batches and receive a dataset with variance independent of absolute expression levels data were renormalized applying vsn2 function (Huber et al., 2002). The log2 intensities for each probe were used for further analysis. Differentially expressed genes were determined applying the limma Bioconductor package and using the functions lmFit and eBayes with default settings. Resulting p-values were adjusted for multiple testing according to Benjamini-Hochberg (Hochberg and Benjamini, 1990). All calculations were performed in the R statistical programming environment (Huber et al., 2002) version 2.8.0. Hierarchical clustering (Eisen et al., 1998) was performed using MeV4.0.01 (Saeed et al., 2003) applying euclidean distance and average linkage.

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Transcription profiling of human colon cells induced to differentiation

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