Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disease of early childhood with both myeloproliferative and myelodysplastic features. We had previously identified the genes for the alpha-chain of the nascent polypeptide-associated complex (NACA) and annexin II (ANX2) as potentially involved in the pathophysiology of JMML. Now we used SMART cDNA synthesis and subsequent "virtual Northern" blot to analyze differential expression of NACA and ANX2 genes in various hematologic cell lines and compared the data to those obtained by standard Northern analyses. The results show that SMART cDNA reproduces the expression profile found in mRNA. Dilution experiments showed that analyses using as little as 0.5 ng of total RNA led to reliable results. After validating the technique, we used virtual Northern blots to analyze expression of NACA and ANX2 in progenitor cultures of nine children with JMML and five healthy individuals. We found no consistent pattern of differential expression between patients and healthy donors. We conclude that aberrant regulation of NACA or ANX2 does not play a relevant role in JMML pathogenesis.