RT real-time PCR (RT-qPCR)

HepG2 cells (2 × 10⁵ cells/well) were infected with HF strain at an MOI of 1 for 30 min and subsequently cultured in the presence (or absence) of MG132 for 20 hours. mRNA was extracted from HSV-1 infected cells using RNAiso Plus (Takara, Osaka, Japan). cDNA was synthesized by Revetra Ace qPCR kit (Toyobo, Oaka, Japan) and subjected to SYBR green real-time PCR⁶². Relative mRNA expression levels were determined by GAPDH expression and ΔΔCt methods. The sequences of oligonucleotides used for RT-qPCR primers were as follows: Us12-forward 5′-AGATCGTAGTGTCCGCACCG-3′, Us12-reverse 5′-CTTAAAAGGCGTGCCGTCCG-3′, UL19-forward 5′-AACAGCCTGTACGACGTC-3′ and UL19-reverse 5′-AACTTGGTACACACGCACGC-3′) The primer set for GAPDH (forward 5′-CATCAAGAAGGTGGTGAAGCAG-3′ and reverse 5′-TGTCGCTGTTGAAGTCAGAGG-3′) was used as an internal control for normalization. For quantification, the expression level of each gene was normalized to that of the GAPDH gene.