Enrichment of genes that are predominantly expressed in hematopoietic stem cells.

(A) A schematic drawing illustrating the strategy of systematic analyses of gene expression in hematopoietic stem cells (HSCs). Bacterial colonies from the 2 HSC cDNA libraries A and B described in the text were spotted in duplicates onto 24 × 24 cm nylon membranes using a Q-bot robotic system (Genetix), generating macroarrays. The macroarray membranes were hybridized with ³³P-labeled cDNA probes derived from mature cell lineages described in the text in parallel with the vector-derived probe. Hybridization signals were analyzed using Spotfinder software (see “Materials and methods” for detailed description) and converted to numeric numbers using Microsoft Excel. cDNA clones, showing negative or very weak hybridization signals to the cDNA probes derived from lineage-positive cells but positive to the vector-derived probe, were selected. This process was called negative selection. The negatively selected cDNA clones were analyzed by DNA sequencing, and spotted in duplicate onto microarray slides. Total RNA isolated from Rh^loLin^−/loc-kit⁺Sca-1⁺or Rh^hiLin^−/loc-kit⁺Sca-1⁺HSCs was used to generate Cy3- and Cy5-labeled cDNA probes, respectively, by RT-PCR. The probes were hybridized to the microarray slides and the data were analyzed using array analysis software. Details of the experiment are described in “Materials and methods.” (B) Northern analysis of unknown genes from cDNA library B. Two micrograms of poly A⁺ RNA purified from indicated tissues was separated on a 1% agarose-formaldehyde gel, transferred to nylon membrane, and then hybridized with a ³²P-labeled probe prepared from each unknown clone. Four representative blots are shown.