The number of genes differentially expressed at three different time points (6, 24 and 72 hpv) of PRRSV vaccination compared to the control (before vaccination)

The number of genes differentially expressed at three different time points (6, 24 and 72 hpv) of PRRSV vaccination compared to the control (before vaccination). pigs. Results Transcriptome profiling of PBMCs from PRRSV vaccinated and age-matched unvaccinated pigs at right before (0?h), and at 6, 24 and 72?h after PRRSV vaccination was performed using the Affymetrix gene chip porcine gene 1.0 st array. Comparison of PBMCs transcriptome profiles between vaccinated and unvaccinated pigs revealed a distinct host innate immune transcriptional response to PRRSV vaccine. There was a significant temporal variation in transcriptional responses of PRRSV vaccine in PBMCs accounting 542, 2,263 and 357 differentially expressed genes (DEGs) at 6, 24 and 72?h post vaccination, respectively compared to the time point before vaccination (controls). Gene ontology analysis revealed the involvement of these DEGs in various biological process including innate immune response, signal transduction, positive regulation of MAP kinase activity, TRIF-dependent toll-like receptor signaling pathway, Deferasirox Fe3+ chelate T cell differentiation and apoptosis. Immune response specific pathways such as cytokine-cytokine receptor interaction, chemokine signaling pathway, signal transduction, JAK-STAT pathway and regulation, TRAF6 mediated induction of NF-kB and MAPK, the NLRP3 inflammasome, endocytosis and interferon signaling were under regulation during the early stage of PRRSV vaccination. Network enrichment analysis revealed and as the highly interconnected hubs of the functional network of PRRSV vaccine induced transcriptome changes in PBMCs. Conclusions This study showed that a massive gene expression change occurred in PBMCs following PRRSV vaccination in German Landrace pigs. Within first 3?days of vaccine exposure, the highest transcript abundance was observed at 24?h after vaccination compared to that of control. Results of this study suggest that and could be considered as potential candidate genes for PRRSV vaccine responsiveness. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2849-1) contains supplementary material, which is available to authorized Deferasirox Fe3+ chelate users. value of a given network module was calculated using a Wilcoxon rank-sum test of the internal (edges Deferasirox Fe3+ chelate within in a module) and external (edges connecting the Deferasirox Fe3+ chelate nodes of other modules) degrees. The p values were calculated based on their connectivity assuming null hypothesis that there is no difference between the number of internal and external connections to a particular node in the module. Module having more internal than external edges was like to be significant. The functional enrichment of modules was performed with REACTOME.db pathway database incorporated in this tool for comprehensive biological illustration of the network. Quantitative real-time PCR (qRT-PCR) For technical validation of microarray results, five selected DEGs (Table?1) known to be involved in immune response function were quantified by qRT-PCR in the same RNA samples as used for microarray expression. Primers were designed based on an open source primer designing software Primer3 [41]. Deferasirox Fe3+ chelate First Strand cDNA Synthesis Kit (P/N?K1612, Thermo Scientific, Co.) was used for reverse transcription with oligo (dT) primer. The qRT-PCR reaction was set up taking 1.0?l of cDNA template, 8.0?l of deionized RNase free water, 0.5?M of upstream and downstream primers, and 10?l iTaq? Universal SYBR? Green Supermix (Bio-Rad laboratories GmbH, Germany) in a total reaction volume of 20?l and were amplified by the StepOnePlus? Real-Time PCR System (Applied Biosystems?, Darmstadt, Germany). The thermal cycling conditions were 95?C for 3?min, 95?C for 15?s, 6?C for 45?s (40?cycles); 95?C for 15?s, 62?C for 1?min, 95?C for 15?s. All reactions were run in duplicate and the average value was used for calculating the expression value. Gene-specific expression was measured as relative to the geometric mean of the expression of two housekeeping genes (GAPDH and ACTB) (Table?1). The delta delta Ct (??Ct) method was used for calculating the difference between target gene and reference genes [42]. The correlation between microarray and qRT-PCR results was analyzed by Spearmans Rho test. The significance level was set as normalized enrichment score, false discovery rate Differential gene expression in PBMCs after PRRSV vaccination To get a comprehensive overview of transcriptional modifications associated with innate immune response, we performed the differential gene expression analysis over three time points (6, 24 and 72 hpv) after vaccination compared to the control (before vaccination). The normalized expression values for only main probes of the chip were included for differential expression?analysis and filtered by the thresholds of FDR 0.01 and LEFTYB log2 fold-change 1.5 or? ?1.5. Using this criterion, 2,453 transcripts were found to be differentially expressed in PBMCs after PRRSV vaccination. Among them, 1087 (44.31?%) gene transcripts could be annotated. A complete list of the differentially expressed genes (DEGs) in PBMCs at three time points following PRRSV vaccination is provided in Additional files 3, 4 and 5. The number DEGs and their direction of expression.