Reverse transcriptase quantitative real-time PCR (qRT-PCR)

To isolate RNA from infected tissues, brains were snap-frozen in liquid nitrogen and RNA was subsequently isolated using a RNeasy Lipid Tissue Midi Kit (Qiagen). RNA was quantified by spectrophotometry and reverse-transcribed with a High-Capacity cDNA Reverse Transcription Kit and random or oligo dT primers. An aliquot of cDNA was used for qRT-PCR; reagents included were the Universal Probe Library system (Roche) with LCMV-specific primers, and FastStart Universal Probe Master Mix on a Mastercycler EP Realplex (Eppendorf) for 40 (2-step) cycles (95°C, 15s; 60°C, 60s). Relative quantification to the control was done using the comparative C_t method. Viral RNA values were obtained from whole brains isolated from perfused mice. The values plotted are the average of at least 3 PCR reactions, and were normalized to PPIB.