The cDNAs were subsequently KU-57788 price amplified and quantified using SYBR Green PCR reagents (RealQ-PCR Master Mix Kit, Ampliqon, Copenhagen, Denmark) according to the manufacturer’s instructions. Briefly, the cDNA for the SOCS1 and β-actin genes were amplified using AmpliTaq Gold DNA polymerase and gene-specific primers

that were designed using Primer Express software (Applied BioSystems). The primers for SOCS1 were 5′-CCC TGG TTG TTG TAG CAG CTT-3′ and 5′-CAA CCC CTG GTT TGT GCA A-3′, and the primers for β-actin (internal control) were 5′-GGC CAA CCG CGA GAA GAT-3′ and 5′-CGT CAC CGG AGT CCA TCA C-3′. Each PCR reaction mixture (final volume 20 μL) contained the following components: 2 μL of cDNA, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 10 μL of 2× SYBR Green PCR Master Mix, and 6 μL of H2O. The PCR reactions were performed in triplicate. The relative quantification values for the target gene expression were calculated from the Ct values, the PCR cycle at which fluorescence from the SYBR Green dye exceeded that of the baseline signal. To calculate ΔCt, Ct values for β-actin cDNA were subtracted from that of the target cDNA. Three ΔCt values for each sample were averaged. To

calculate the fold-change of SOCS1 expression in cells treated with the miR-150 mimic relative to that of control cells, the average ΔCt value calculated for the control cells was subtracted from that calculated for the miR-150 transfected cells, generating the ΔΔCt value. Next, the fold-change for each well was calculated using click here the 2−ΔΔCt formula. The fold-change values from three wells were Ergoloid averaged.19 and 22 Plasma levels of IFN-α, IFN-γ, IL-10, and IL-13 were measured using enzyme-linked immunosorbent assay (ELISA) kits manufactured by Bender MedSystems Inc., Vienna, Austria. The results were calculated from the interpolation of a standard curve made from a series of known concentrations of commercial standards. DENV-2 (New Guinea C strain) was propagated in Aedes albopictus C6/36 cells.

Virus titres were determined by a standard plaque-forming assay using BHK-21 cells. Titres were adjusted to 2 × 107 PFU/ml in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) containing 10% foetal calf serum (Gibco BRL) in a large-scale preparation. Viruses were collected and stored at −80 °C before use. Human PBMCs were prepared and collected from whole-blood donated from healthy, seronegative, consenting donors by using a DENV antibody detection kit (Gene Labs Diagnostics, Singapore). Blood samples were mixed with 4.5% dextran to separate leukocytes from red blood cells. PBMCs were separated from polymorphonuclear cells by using Ficoll–Paque density centrifugation, and were resuspended to a concentration of 2 × 106 cells/mL.