Quantitative RT-PCR for FCV Detection

The protocol for real-time RT-PCR has been described previously [35 (link)]. Total RNA from drug-treated or untreated cells was isolated using an RNeasy^® Mini Kit (QIAGEN) according to the manufacturer’s instructions. Transcription of RNA into cDNA was performed using a PrimeScript ™ 1st Strand cDNA Kit (Takara, Japan). The following primers for FCV Pro-Pol and GAPDH were designed: FCV-for, 5’-ATGATTTGGGGTTGTGATGT-3’; FCV-rev, 5’-TGGGGCTRTCCATGTTGAT-3’; GAPDH-for, 5’-TGACCACAGTCCATGCCATC-3’; GAPDH-rev, 5’-GCCAGTGAGCTTCCCGTTCA-3’. The procedure for PCR consisted of an initial step at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 15 s. The relative level of RNA expression was determined by the 2^-∆∆CT method [41 (link)]. GAPDH mRNA was analyzed as a loading control.