Fam labeled primers

The total RNA was isolated using The High Pure RNA Isolation Kit (Qiagen). Quality and quantity of the total RNA were verified by the Agilent 2100 Bioanalyzer electrophoresis. The total RNA was used to synthesize complementary DNA by extension of oligo d(T)15 primers with M-MLV reverse transcriptase (Sigma-Aldrich). The Taq-Man ® Array Human Cyclins and Cell Cycle Regulation (contains 44 different genes and 4 endogenous controls) and The TaqMan ® Array Human JAK-STAT Pathway (contains 92 different genes and 4 endogenous) from Applied Biosystems (Germany) were employed. PCR amplifications were performed in triplicate in a 10 μl of reaction volume. For validation of the results of microarrays and TaqMan ® Array profilers, FAM-labeled primers from Applied Biosystems and 2x TaqMan PCR Master Mix were used. For evaluation of STAT3 silencing primers: 5′-ATTGCCCG GATTGTGGCCCG-3′, 5′-CTCCGTCACCACGGCTGCT G-3′, and 2x SybrGreen PCR MasterMix were used. The amount of target mRNA was first normalized to the 18S RNA expression level in the same sample and then to an untreated control. Real-time PCR amplifications were performed in duplicate on complementary DNA equivalent to 25 ng of RNA in 20 µl of reaction mixture containing 2X SybrGreen PCR Master Mix (Applied Biosystems, Darmstadt, Germany).