RNA Extraction and RT-PCR for Viral Load Quantification

Total RNA was extracted from the frozen brain stem using the High Pure RNA Tissue Kit (Roche Molecular Biochemicals, GmbH, Mannheim, Germany). Approximately 30 mg of tissue was homogenized using a homogenization pestle, and RNA was extracted according to the manufacturer’s recommendations. Then, 50 μL of RNA was eluted and stored at −30°C in a low-temperature freezer until further use. For real-time RT-PCR, the LN34 assay was performed using AgPath-ID One-step RT-PCR Reagents (Applied Biosystems, Foster City, CA, USA) [41 (link)–43 ]. The master mix consisted of the following: 6.5 μL of ddH₂O, 12.5 μL of 2× RT buffer, 1 μL of 25× RT-PCR Enzyme Mix, 1 μL of either LN34 or beta-actin primer sets (10 μM), 1 μL of either LN34 or beta-actin probe (5 μM), and 2 μL of RNA template [41 (link)–43 ]. The sealed plate was placed into an ABI Step One Plus Real-Time PCR (Applied Biosystems Foster City, CA, USA), and the following conditions were set: reverse transcription at 50°C for 30 min, denaturation at 95°C for 10 min, and amplification of 45 cycles at 94°C for 15 s and 56°C for 30 s using ABI7500-standard mode. To estimate viral load, the Cq values were divided into >25 (low copy numbers), 15–25 (high copy numbers), and <15 (very high copy numbers).