Total RNA was extracted from PB or BM (patients no. 9 and 10) MNCs using the TRIzol Reagent (Life Technologies, Milan, Italy), according to the manufacturer's instructions. RT was performed on 2 μg total RNA using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase and oligo dT_(12-18) as primer. PCR was performed on 100 ng retrotranscribed total RNA, using 50 pmol each sense and antisense primer. Oligonucleotide primer sequences and gene-specific PCR amplification programs used have been previously reported.⁵  The integrity of RNA and oligo(dT)-synthesized cDNA was confirmed by amplification of all cDNA samples with β-actin-specific primers. Then 10 μL each amplified product was run on a 2% agarose gel and visualized by ethidium bromide staining.

T indicates time from the beginning of decitabine therapy (days); RAEB-t, refractory anemia with excess blasts in leukemic transformation; CMMoL, chronic myelomonocytic leukemia; and nd, not determined.

Secondary AMLs evolved from a previous myeloproliferative disorder.

Figures refer to the percentage of PB atypical monocytes.