CD34+ hemopoietic progenitors purified from peripheral blood.
Growth protocol
Highly purified CD34+ cells from 6 healthy donors were seeded at 1000000 cells/ml in serum free medium (EX vivo 15) w/o cytokines and treated with 10 mM UTP for 1 hour.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated 1000000 cells using a modification of the guanidinium isothiocyanate procedure and ultracentrifugation on cesium chloride gradient. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 bioanalyzer. RNAs originating from the 6 donors were pooled in order to obtain at least 2ug per sample.
Label
biotin-labeled
Description
The biotin-labeled target synthesis reactions, as well as the Affymetrix HG-U133A GeneChip arrays hybridization, staining, and scanning, were performed, starting from 2ug of total cellular RNA, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA). The fragmented cRNAs (20ug) were then hybridized to an identical lot of Affymetrix HG-U133A GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v4 protocol, using antibody-mediated signal amplification. GeneChip were finally scanned using the GA2500 Affymetrix GeneChip scanner.
