Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Transcriptional dysregulation mediated by RUNX1-RUNX1T1 in normal human progenitor cells and in acute myeloid leukaemia

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In order to identify genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression during myeloid and erythroid development of normal human progenitor cells.

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Transcription profiling of human normal progenitor cells mutated for RUNX1-RUNX1 during myeloid and erythroid development to identify genes disregulated by RUNX1-RUNX1

In order to identify genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression during myeloid and erythroid development of normal human progenitor cells.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-20787

Neonatal cord blood was obtained from normal full-term pregnancies at the Maternity Unit of the University Hospital of Wales (UHW), Cardiff, U.K. Human CD34+ cells (>95% pure) were derived from cord blood using MiniMACS (Miltenyi Biotec, Surrey, U.K.) according to the manufacturer's instructions. These cells were subsequently cultured overnight at 1x10^5 cells/ml in IMDM containing 20% FCS and the following growth factors (from R&D Systems, Abingdon, U.K.): IL 3 (5 ng/ml), IL 6 (10 ng/ml), SCF (20 ng/ml), GM CSF (5 ng/ml), G CSF (5 ng/ml), Flt3 ligand (5 ng/ml) and on the following two days retrovirally transduced as previously described (Tonks 2004, leukaemia). In this way RUNX1-RUNX1T1 (RR) and control matched transduced cells were derived on day 3.   These cultures were also grown until day 6 and were fractionated into the main progenitor groups for microarray analysis. A depletion strategy using the Miltenyi Biotec MiniMACS system was used for this purpose. Briefly, myeloblasts (CD15+) were enriched from mixed lineage transduced cells by serial depletion of monoblasts (CD14hi) and erythroblasts (CD36+) using immunomagnetic beads directed against those antigens.

Transcription profiling of human normal progenitor cells mutated for RUNX1-RUNX1 during myeloid and erythroid development to identify genes disregulated by RUNX1-RUNX1

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