The reaction mix contained 1 iQSupermix, 200 new nM of each primer, 400 nM of fluorescent probe and 250 ng of DNA. The analyses were performed using a C1000 Touch Thermal cycler. All real time TaqMan PCRs for CyHV 3 DNA were run with equal amounts of DNA estimated by the real time TaqMan PCR performed on carp glucokinase gene. Quantification of carp gene expression in spleen by RT qPCR Total RNA was isolated from spleens stored at ?80 C in RNALater using TRI reagent, including DNase I digestion and RNA purification using RNeasyMinElute Cleanup Kit. cDNA was synthetized from 1 ug of RNA using iScriptcDNA Synthesis Kit. The primers used for RT qPCR were described previously and are listed in Table 1. The RT qPCR master mix was prepared as follows 1 IQ SYBR Green Supermix, 200 nM of each primer, 5 uL of 25 diluted cDNA and sterile water to a final volume of 25 uL.
The amplifica tion program included an initial denaturation at 95 C for 10 min, followed by 40 Inhibitors,Modulators,Libraries cycles with Inhibitors,Modulators,Libraries denaturation at 95 C for 15 s, annealing at 58 C for 30 s and elongation at 72 C for 30 s. At the end, the dissociation stage was performed and the melt curve was obtained by in creasing Inhibitors,Modulators,Libraries the temperature from 60 C to 95 C with a rate of 0. 5 C per 5 s. Fluorescence data from RT qPCR experi ments were analyzed using the CFX96 real time system and exported to Microsoft Excel. The threshold cycle was determined using the Auto method for all runs. The expression of analyzed genes was calculated using the 2 Ct method. The 40S ribosomal protein S11 was used as a reference gene.
Histological analysis Organs from mock infected or infected carp were fixed in 4% buffered formalin and embedded Inhibitors,Modulators,Libraries in paraffin blocks. Sections of 5 um were stained with haematoxylin and eosin prior to microscopic Inhibitors,Modulators,Libraries analysis. Statistical analyses Multi step growth curves data expressed as mean titer standard deviation were analyzed for significance of differences using one way ANOVA. The differ ences in mortality induced by the CyHV 3 strains tested were analyzed using Kaplan and Meier survival selleck chem Nutlin-3a analysis. Significant differences in virus load between fish infected with the different CyHV 3 strains at each sampling point were assessed using one way ANOVA followed by Holm Sidak test when data were normally distributed, or with the non parametric Kruskal Wallis test followed by Tukey test when they were not.