Human cDNA expression arrays in normal and malignant monocytes

New technologies have recently become available with complementary DNA (cDNA) microarrays, which allow simultaneous analysis of multiple cell gene expression. In hematological malignancies few data have been reported so far. We decided to investigate the usefulness of this challenging methodology in combination with immunomagnetic cell separation techniques. For that purpose, we performed a series of consecutive microarrays on different cell populations. Our samples consisted of monocytes from healthy donors, AMML and CMML patients. As reference we used cells from the KBM3 monocytic leukemia cell line. Cell separations based both on negative and positive selection were tested, in order to verify possibility of gene activation or suppression elicited by the interaction of monoclonal antibodies and surface antigens. After density gradient-based mononuclear cell enrichment, CD14+ cells were sorted using the MACS recommended procedures. FACS analysis was performed on separation products to test the purity of monocytic populations. Both mRNA and total RNA were tested for some of the samples, 32P-radiolabeled cDNA was synthesized and hybridized to Atlas Human cDNA expression array (Clontech) encompassing 588 selected genes. We were able to identify some major recurrent differences between normal and CMML monocytes. Results obtained with mRNA and total RNA were comparable. Negatively-isolated CMML monocytes showed an overexpression of chemokine genes such as migration inhibitory factor-related proteins 8 (MRP8) and 14 (MRP14) and a downregulation of the interleukin-13 (IL-13) precursor gene. Interestingly, low level of expression of some of these genes appears to be upregulated by positive selection performed by CD14-conjugated microbeads in normal monocytes, while no changes were observed in negatively selected monocytes. While human cDNA expression arrays seem to represent a reliable tool for the study of gene expression profiles, we experience some variability between different patients. In conclusion, our results demonstrate that cell separation methodology has an impact in the expression of certain genes and must be taken into consideration. Besides a statistical evaluation of the results, strict controls are always recommended.