Transcription profiling of mouse erythroleukaemic cell line after DSMO-induced differentiation

ABSTRACT: 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 manufacturer’s 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 Tukey’s 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.

ORGANISM(S): Mus musculus

SUBMITTER: Jan Christian Bryne 

PROVIDER: E-MEXP-2454 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress