age: 47 tissue: bone marrow karyotype: healthy control
Treatment protocol
primary cells
Growth protocol
primary cells
Extracted molecule
total RNA
Extraction protocol
Human bone marrow mononuclear cells were enriched for CD34 expression using Miltenyi MACS technology. Afterwards, cells were stained with antibodies against lineage-antigens (CD2, CD3,CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20 CD56, Glycophorin A), CD34, CD38, CD90, as well as CD123 and CD45R in order to distinguish LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), CMP (Lin-/CD34+/CD38+/CD123+/CD45R-), GMP (Lin-/CD34+/CD38+/CD123+/CD45R+) and MEP (Lin-/CD34+/CD38+/CD123-/CD45R-). Cells were subjected to 7-color 5 way sorting utilizing a high-speed cell sorter as previously described (Steidl et al., Nat Genet 2006). Total RNAwas extracted using a denaturing buffer containing guanidine isothiocyanate (ALLprep Kit, Qiagen) from sorted cell populations.
Label
biotin
Label protocol
10 ng of total RNA were used for linear amplification of cDNA using the Ovation Pico RNA Amplification System (Nugen), according to the manufacturer's instructions. 5 µg of cRNA were biotin-labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen).
Hybridization protocol
Following fragmentation, labeled cRNA of each individual sample was hybridized to Affymetrix Affymetrix Human Gene 1.0 ST microarrays (Affymetrix) and stained according to the manufacturer's instructions.
Scan protocol
Arrays were scanned according to the manufacturer's instructions.
Description
C0111N(GMP)
Data processing
Data were normalized normalized with the Expression Console Software version 1.1 from Affymetrix using Robust Multi-array Average (RMA) algorithm. MEV software was used to select differentially expressed genes by applying the following criteria: (a) Absolute value of the group mean difference greater than 1.5, and (b) p value smaller than 0.05 (Welch's t test)
Expression data from human hematopoietic stem and progenitor compartments from patients with acute myeloid leukemia with normal karyotype and healthy controls
