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Status |
Public on Jan 01, 2013 |
Title |
RB585-NN |
Sample type |
RNA |
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Source name |
CML CD34+
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Organism |
Homo sapiens |
Characteristics |
tissue: bone marrow msc treatment: No MSC imatinib treatment: No IM
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Treatment protocol |
CML CD34+ cells were treated with or without IM (5μM) in the presence or absence of MSC for 96 hours (n=3 each).
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Growth protocol |
CML CD34+ cells were cultured in the presence or absence of irradiated MSC at 37°C with 5% CO2 in serum-free expansion medium (SFEM, Stem Cell Technologies, Vancouver, BC,) supplemented with GFs at concentrations similar to those found in stroma-conditioned medium from long-term bone marrow cultures [granulocyte-macrophage colony-stimulating factor (200pg/mL), granulocyte colony-stimulating factor (1ng/mL), stem cell factor (200pg/mL), leukemia inhibitory factor (50pg/mL), macrophage inflammatory protein-1 (200pg/mL), and interleukin-6 (1ng/mL)].
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted using the RNAeasy Mini Kit (Qiagen, Valencia, CA), amplified, labeled and hybridized to GeneChip 1.0 arrays (Affymetrix, Santa Clara, CA).
|
Label |
biotin
|
Label protocol |
100ng total RNA for each sample was linear amplified and labeled with Biotin
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Hybridization protocol |
~5.5ug of labeled cDNA was hybridized to Affymetrix Gene Array ST-1.0
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Scan protocol |
Arrays were scanned at 5um resolution with Affymetrix scanner 3000 7G
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Description |
Gene expression data from CML CD34+ cells
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Data processing |
Microarray data analysis was performed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). The 33297 probes represented on the microarray were filtered by cross-sample mean, and for standard deviation of greater than the 25% quantile, yielding 18624 probes representing 12553 genes. Linear regression was used to model the gene expression with the consideration of a 2x2 factorial design and matched samples. Differentially expressed genes were identified by calculating empirical Bayes moderated t-statistic, and p-values were adjusted by FDR using the “LIMMA” package. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software version 2.04 [http://www.broadinstitute.org/gsea/] to detect enrichment of predetermined gene sets using t-scores from all genes for 1263 gene sets in the C2 (curated gene sets) category from the Molecular Signature Database (MsigDB)41. For significant gene sets related to the Wnt/β-catenin and Cadherin pathways, leading edge genes commonly enriched in these sets were analyzed. Heatmaps were plotted to show contrasts amongst the different treatment groups.
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Submission date |
Dec 31, 2012 |
Last update date |
Jan 01, 2013 |
Contact name |
Ravi Bhatia |
E-mail(s) |
rbhatia@coh.org
|
Phone |
6263598111
|
Fax |
6263018973
|
Organization name |
City of Hope Beckman Research Institute
|
Department |
Hematopoietic Stem Cell and Leukemia Research
|
Street address |
1500 E Duarte Road
|
City |
Duarte |
State/province |
CA |
ZIP/Postal code |
91010 |
Country |
USA |
|
|
Platform ID |
GPL6244 |
Series (1) |
GSE43225 |
The effect of IM and MSC treatment on gene expression in CML CD34+ cells |
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