For HSC and HPC isolation, c-Kit positive cells were first enriched by immuno-selection with CD117 micromagnetic beads (Miltenyi Biotec, Auburn, CA). Cells were then stained with FITC-conjugated anti-CD34, PE-conjugated anti-Sca-1, allophycocyanin (APC)-conjugated anti-c-Kit, and a PE-Cy7-conjugated lineage marker cocktail consisting of anti-Gr-1, CD11b, B220, CD3, CD4, CD8, and Ter119 monoclonal antibodies (BD PharMingen, San Diego, CA, and eBioscience, San Diego, CA). 4ยด,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) was added for dead cell discrimination. Based on multi-color flow cytometric analysis, LT-HSC (CD34-LKS), ST-HSC (CD34+LKS) and HPC (LKS-) were sorted by using a MoFlo High-Speed Cell Sorter (Dakocytomation, Fort Collins, CO).
Extracted molecule
total RNA
Extraction protocol
Cells were thawed, selected based on morphology, and 50 or 100 were packed into empty, washed zonae pellucidae for ease with handling. Individual cell-packed zonae pellucidae were transferred to 1.5 ml microcentrifuge tubes and snap frozen on liquid nitrogen and then stored at -80 ?C. Total RNA was extracted from 50-200 cells from each cell type using TRIzol reagent (Invitrogen, Carlsbad, CA) with linear acrylamide (Ambion, Inc., Austin, TX) as a co-precipitant.
The isolated RNA immediately underwent linear amplification using the TargetAmp? 2-Round Aminoallyl-aRNA Amplification kit 1.0 (Epicentre Biotechnologies, Madison, WI). After amplification, the aminoallyl-aRNA was cleaned using an RNeasy kit (Qiagen, Venlo, The Netherlands) and concentration was determined by absorbance at 260 nm. Aminoallyl-aRNA was quick frozen in a dry ice ethanol bath and stored at -80 ?C until labeled with biotin-X-X-NHS (Epicentre Biotechnologies). The labeling reaction was cleaned using the Minelute cleanup kit (Qiagen) and the concentration was determined.
Hybridization protocol
10 ?g of labeled aRNA from each cell type was hybridized to an Affymetrix? Mouse 430-2 GeneChip. Fragmentation of aRNA, hybridization, staining, and scanning of the GeneChip was carried out according to the manufacturer's protocol.
