Study of gene expression following Pim2 knockdown in FLT3-ITD-positive acute myeloid leukemia

The objective of the sudy is to explore the biological pathways controlled by Pim2 in FLT3-ITD positive AML We used microarrays to investigate gene expression in the MOLM-14 AML cell line in which Pim2 knockdown was induced by RNA interference (doxycycline-inducible shRNA) MOLM-14 cells transduced by doxycycline-inducible scrambled or anti-Pim2 shRNA were cultured 72h in the presence of 200 ng/ml doxycycline. Total RNA was extracted with DNase I digestion (RNeasy Mini Kit used with the Bio-robot QIAcube). Biotinylated cDNAs were prepared from 100ng total RNA using the GeneChip® Whole Transcript Sense Target Labeling Assay Kit according to the standard Affymetrix protocol (Affymetrix, Santa Clara, CA, USA). Following fragmentation, 10ug of cDNA were hybridized for 16h at 45°C on GeneChip® Human Gene 2.0 ST arrays arrays (Affymetrix), washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the GCS 3000 scanner (Affymetrix).