Temporal Expression of Viral Transcripts

The real-time PCR was performed on the 1st, 3rd, 7th, 14th, and 21st days after intrathecal injection of the virus. Total RNA (6 samples of each group) was extracted from L4-L5 spinal cord. Extracted RNA was treated with DNase I at 37°C for 30 min before reverse transcription was performed using a kit (TaKaRa, Japan). The PCR primers were as follows: 5′-CGGGAG CTC TGA ATG CTC TCT TGC ATC TGG CTG GC-3′ (forward) and 5′-CGG GTC GAC GCC ATA CAA TTC GACCTG CTG-3′ (reverse). The Real-Time PCR Detection System (Roche, Switzerland) continually monitors the increase in fluorescence, which is directly proportional to the PCR product [15 (link)].