qRT-PCR analysis

For quantitative analysis of changes in transcription from brain tissue using qRT-PCR arrays, 400 ng of high-quality RNA from each sample was reverse transcribed to synthesize cDNA using the RT² First Stand Kit per manufacturer’s instructions (QIAGEN). For quantitative analysis in transcription from adult microglia, 10 ng of RNA from each culture well was pre-amplified using the RT² PreAMP cDNA synthesis kit (330451) with accompanying RT² PreAMP Mouse Inflammatory Cytokine & Receptors Pathway Primer Mix (330241 PBM-011Z) according to instructions by QIAGEN. Each cDNA reaction was mixed with 2x RT2 SYBR Green Mastermix purchased from QIAGEN with RNase-free water to a final volume of 1.3 ml. Ten microliters of the mixture was then added to each well of a 384-well format plate of either the Mouse Inflammatory Cytokine & Receptors super array PAMM-011ZE or Mouse Toll-Like Receptor Signaling super array PAMM-018ZE (QIAGEN).

The analysis was carried out on an Applied Biosystems ViiA 7 Real-Time PCR System with a 384-well block using the following conditions: 1 cycle at 10 min, 95 °C; 40 cycles at 15 s, 95 °C then 1 min, 60 °C with fluorescence data collection. Melting curves were generated at the end of the completed run to determine the quality of the reaction products. Raw threshold cycle (C_T) data was collected with a C_T of 35 as the cutoff. C_T data was analyzed using the web-based RT² Profiler PCR Array Data Analysis from QIAGEN. All C_T values were normalized to the average of the C_T values for the housekeeping genes Actb, Gapdh, and Hsp90ab1. Changes in transcription were calculated by the software using the ΔΔC_T based method [46]. Statistical analysis was performed using the unpaired Student’s t test to compare the replicate ΔC_T values for each gene in the control group versus infected groups [47]. A mean of ≥ or ≤ 2.0-fold change and P value of ≤ 0.05 were considered significant. For qRT-PCR data, we did not adjust P values for multiple comparisons since we were interested in only controlling for the individual error rate, where an adjustment for multiple tests is deemed unnecessary. Venn diagrams and heatmap hierarchical cluster analysis of the full datasets were composed using InteractiVenn [48] and Heatmapper [49], respectively.