18 and 19 Blood samples were drawn from the study participants between 1 and 3 day after individuals were admitted

to the Kaohsiung Chang Gung Memorial Hospital. We obtained blood samples from one patient with DHF, from the same number of patients with classic DF, from those with other non-dengue febrile SB431542 illness (OFI, presumed to be viral illness). Forty-one RNA samples from patients without or with confirmed DENV-2 infection (15 DF, 14 DHF, and 12 OFI patients) were reverse-transcribed into cDNA. Using these cDNA samples, we investigated whether SOCS1 expression levels correlated with the severity of DF and the expression of its regulatory miRNA. DENV-2 infection was confirmed by the presence of clinical dengue symptoms, the detection of DENV-2 RNA by using quantitative RT-PCR in the blood samples. As we previously described, the diagnosis of DHF was made according to the criteria of the World Health Organization, which included the presence of thrombocytopaenia

(<100,000/mm3), haemorrhage, and evidence Target Selective Inhibitor Library solubility dmso of plasma leakage, as indicated by haemoconcentration (≥20%), pleural effusion, ascites, and/or hypoalbuminaemia.20 and 21 The OFI patients were identified as those who had a fever but no detectable DENV-specific immunoglobulin M or DENV RNA in leukocytes, and no obvious bacterial aetiology for their illness. Thus, these patients were presumed to have a non-dengue viral illness.20 and 21 Total RNA was isolated from peripheral blood mononuclear cells

(PBMCs) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Stem-loop RT-PCR analysis of miRNA expression was performed as described previously.22 All reagents were obtained from Applied BioSystems (Foster City, CA, USA). Briefly, 50 ng of total RNA was reverse-transcribed into cDNA by using stem-loop primers and the TaqMan microRNA Reverse Transcription kit. miRNA expression was quantified using the Applied BioSystems 7500 Real-time PCR pheromone System and the TaqMan Universal PCR Master Mix. We searched for miRNA-targeted genes in an online public database system, including miRBase Targets (http://microrna.sanger.ac.uk/), and TargetScan (http://www.targetscan.org) data analysis.23 Eleven miRNAs (miR-15a, miR-20, miR-21, miR-96, miR-126, miR-146, miR-150, miR-181a, miR-155, miR-221, and miR-572) were identified as potential regulators of SOCS1 expression (Fig. 3(a)). To compare the levels of miRNA expression, we normalised their expression to that of the internal control 5S rRNA. Comparative threshold (Ct) values were used to calculate the relative miRNA expression. The amount of each miRNA relative to 5S rRNA was calculated using the equation 2−ΔCt, where ΔCt = (CtmiRNA − Ct5S). The PCR reactions were run at 95 °C for 15 min followed by 40 cycles of denaturing at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. All reactions were performed in triplicate.