Inhibition of Jak2 in SET2 cell line with INC424 (two time points and 3 dose levels in triplicate)

SET2 cells were incubated with increasing concentration of INC424/Ruxolitinib for 3-6 h and RNA was purified. Affymetrix Genechip miRNA array 2.0 was used to identify miRNA species showing a differential expression in treated cells compared with control cells. Exponentially growing SET2 cells were cultured in medium supplemented with 10% fetal bovine serum in the presence or the absence (control cells) of 160, 800 e 1,600 nM concentrations of the JAK1 and JAK2 inhibitor INC424/Ruxolitinib for 3 and 6 h. Triplicate cultures were prepared for each drug concentration and culture time. Total cellular RNA, including small RNAs, was extracted using RNeasy Micro kit (Qiagen, Valencia, CA). Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA. Total RNA (500ng for samples) was labeled using the FlashTag Biotin HSR kit (Genisphere) and hybridized to the Affymetrix Genechip miRNA array 2.0 using manufacturer's protocols.