Pax5 expression in circulating CD34⁺sIg⁻subsets correlates with CD19 but not with CD10 surface expression.

Surface IgM⁻CD34⁺CD19⁻CD10⁻, IgM⁻CD34⁺CD19⁻CD10⁺, IgM⁻CD34⁺CD19⁺CD10⁻, and IgM⁻CD34⁺CD19⁺ CD10⁺sorted single cells were analyzed for mRNA expression ofPax5 after multiplex RT-PCR. For simplicity, the results for GAPDH, VpreB, TdT, RAG-1, and CD79a genes, covered in Figure 2 and Table 1, are not included. In each of the 4 IgM⁻CD34⁺ subsets, 19 of 20 (95%) tracks showed a neat GAPDH⁺ amplification. RT-PCR tubes receiving one FACS droplet were labeled numbers 1 to 20. Similarly processed positive controls (+) corresponded to individual Nalm-6 pre-B leukemia single cells, whereas all reagents without cells were added in the negative controls (−). Results are representative of 5 similar experiments that analyzed the 4 CB populations from independent donors. Approximately 80% of the cells showed neat Pax5 mRNA amplifications in the IgM⁻CD34⁺CD19⁺CD10^+/−subset, but less than 5% (less than 1 of 19) cells in the CD34⁺CD19⁻CD10^+/− subsets bore Pax-5 mRNA. RT-PCR fidelity was ascertained by sequencing of the cDNA in the excised bands and by molecular weight estimation. L indicates ladder.