Gene expression arrays and statistical analysis

Spleens were harvested from rats at approximately 8 weeks post-infection and a portion flash frozen on dry ice. Samples were stored at −80°C until RNA extraction using the RNeasy RNA kit (Qiagen). Purified RNA was treated with Ambion Turbo Free DNase (Thermo-Fisher) and sample quality checked using the BioAnalyzer 2,100–RNA Chip Nano 6,000 (Agilent, CA). Two microgram of RNA was used per 150 μl cDNA synthesis reaction (Invitrogen Superscript IV First Strand Synthesis kit) for each animal. cDNA amounts were normalized and pooled such that 20 ng from three infected or non-infected animals provided 60 ng per each well of a BioRad Prime PCR custom array PCR assay plate. Genes assayed, relevant gene identification information, and primer sequence as supplied by the manufacturer are supplied in Supplementary Table 2. RT-PCR assays were performed using SSOAdvanced Universal SYBR Green Supermix (BioRad) on 384 CFX C1000 Touch Thermal Cycler (BioRad). QRT-PCR analysis was carried out using BioRad CFX Manager Software using Single Threshold and the relative gene expression levels were calculated using comparative Ct (ΔΔCt) method, normalized to the expression of 2 housekeeper genes (actb, b2m). Replicate wells with Ct standard deviations >0.5 were removed from further analysis. Gene expression was calculated as 2^−ΔΔCt and then Log₂ transformed using GraphPad Prism 7 for statistical analysis. Statistical significance was determined by multiple t-tests using the Holm-Sidak method with alpha = 0.05. An expression ratio of 1.5 was chosen as difference from control (non-infected).