Real-Time Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted using Trizol reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. The total RNA (1 μg) was reverse-transcribed into cDNA in a 20-μl reaction mixture containing 2.5 μM of random hexamers, 2 μM of deoxynucleotide triphosphates, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). We performed PCR in a 20-μl reaction mixture containing 0.2 μl of cDNA, 0.5 μM of forward and reverse primers, and 10 μl of Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7300 real-time cycler (Applied Biosystems). Relative mRNA levels were determined using the 2^−ΔΔCt method, as described in the Applied Biosystems User Bulletin No. 2 (P/N 4303859). The primer sequences for PCR are listed in Table 1 .

Primers used for real-time qRT-PCR.

NP, nuclear protein; UL, unique long gene; NS, non-structural protein.