Computational protocol: Live attenuated African swine fever viruses as ideal tools to dissect the mechanisms involved in viral pathogenesis and immune protection

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Protocol publication

[…] After necropsy, gastrohepatic lymph node samples were aseptically collected, overnight treated with RNAlater® (Ambion, Austin, TX, USA) at 4 °C to increase the RNA stability of the tissues and next frozen at −80 °C for further RNA isolation. RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA, USA), treated with RNase-free DNase Set (Qiagen, Valencia, CA, USA) and impurities removed with RNeasy Minielute kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. The integrity, quality and quantity of RNA obtained was checked in 1% (w/v) agarose gels and using a NanoDrop™ 1000 Spectrophotometer (Thermo Scientific, Rockford, IL, USA). qPCR technology was used to determine the relative expression of a panel of 19 swine gene transcripts representative of Th1, Th2 and Th17 responses (Table ), previously described [, ]. One μg of RNA isolated from infected and control animals at different times post-infection was reverse transcribed to cDNA using the iScript cDNA Synthesis kit (BioRad, Hercules, CA, USA). Three animals from each time point and group were selected taking into account the quality and quantity of the RNA obtained. A PCR specific for the 18S was performed with all cDNAS in order to assess their quality. Final concentration of primers in the PCR reactions was 0.5 μM and primers (Table ) were designed using Beacon Designer™ (Biosoft International, Palo Alto, CA, USA) and internal normalization of the gene expression analysis was carried out by using beta-actin and PPIA as reference genes []. The relative gene expression was assessed by the 2−ΔΔCt method [] and applied as previously described []. With this method, a fold-change value of 1 or −1 represents no difference in gene expression. The differences in mRNA expression among groups were assessed by the Student’s paired T-test using SigmaPlot v10.0 (Systat Software Inc., CA, USA). Values of p < 0.05 were considered as being significant. […]

Pipeline specifications

Software tools Beacon Designer, SigmaPlot
Applications Miscellaneous, qPCR
Organisms African swine fever virus
Diseases African Swine Fever