RNA (3 μg) was reverse-transcribed Selleck RG7422 using oligo (dT) 15 primer (Promega) following manufacturer’s instructions. Primers used for the amplification of hamster

Prdx6 cDNA were 5′-ACTTTGAGGCCAATACCAC-3′ and 5′-TGTAAGCATTGATGTCCTTG-3′, and for GAPDH cDNA 5′-AGAAGACTGTGGATGGCCCC-3′ and 5′-TGACCTTGCCCACAGCCTT-3′ (based on GenBank Accession nos NM177256.3 for Prdx6 and DQ403054.1 for GAPDH). To confirm the identity of the PCR product, amplicon was cloned and sequenced (18). Sequence of amplicon and per cent identities with rat, mouse and human homologous sequences are shown in Table 1. Relative mRNA expression was determined using an ABI7500 thermal cycler by SYBR Green assay (19). Each experiment was performed Small molecule library high throughput in triplicate. In brief, PCR reaction solution (20 μL) contained 5 μL of cDNA, PCR buffer, 0·25 mm of each dNTP, 5 pmol of each primer, 0·5X SYBR Green and 1 U of Hot start Taq polymerase (MBI Fermantas, St. Leon-Rot, Germany). PCR thermocycling conditions were 95°C for 10 min, followed by 40

cycles of 95°C for 15 s and 55°C for 30 s and 72°C for 1 min. A dissociation curve was constructed in the range of 60–99°C. All data were analysed using the Rotor Gene 5 software (Corbett, Australia) with a cycle threshold (Ct) in the linear range of amplification and then processed by the method relative to GAPDH mRNA (20). Localization of Prdx6 expression in O. viverrini-infected hamster liver was performed by an immunohistochemical procedure (19). In brief, paraffin sections (5-μm thick) were deparaffinized in xylene and rehydrated in descending gradations of ethanol. To enhance immunostaining, sections were placed in citrate buffer (pH 6·0) and autoclaved at 120°C for 10 min for antigen unmasking. Tissue sections were incubated overnight with rabbit polyclonal anti-Prdx6 antibody (1 : 100; Abcam) at room temperature. Sections were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG antibody (1 : 200; Zymed Laboratory) and stained sections were Arachidonate 15-lipoxygenase visualized with 3,3-diaminobenzidine tetrahydrochloride

as a chromogen and haematoxylin was used for counterstaining. Data are presented as means ± SD. The Student’s t-test was used to compare between uninfected and infected groups. Statistical analysis was performed using the SPSS version 11·5, with P value <0·05 considered as significant. Differential patterns of protein expression in hamster livers were identified by two-dimensional gel electrophoresis (2DE) (Figures 1 and 2). On the average, 380–400 protein spots were detected in O. viverrini-infected and uninfected groups. Among them, 250–350 protein spots were successfully matched between infected and uninfected groups, with expression levels of 49 proteins being significantly different between these groups (P < 0·05) (Table 2). O.