2.9. Cytokine mRNA Quantification by qRT-PCR

MRC-5 cells (1 × 10⁵) were seeded in the wells of 24-well tissue culture plates. The next day, the cells were infected with HCoV-OC43, and each compound (5 μM) was added to the wells. Total RNA was isolated from MRC-5 cells at the designated time using the RNeasy^® Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was synthesized to complementary DNA using the One Step SYBR® PrimeScriptTM RT-PCR Kit (Takara Bio, Inc., Shiga, Japan) according to the manufacturer’s instructions. The sequences of the human-specific primers used for qRT-PCR are listed in Table 1.

Sequences of the human-specific primers used for qRT-PCR.