Thunderbird probe one step rt qpcr kit

The total RNA was extracted from the inoculated mouse cells or lung tissues using TRIzol Reagent (Thermo Fisher Scientific) and RNeasy Mini Kit (QIAGEN). The extracted RNAs were subjected to quantification using qRT‐PCR analysis; with the THUNDERBIRD Probe One‐step RT‐qPCR Kit (TOYOBO). A primer probe sets for N2 (Takara Bio) was used. The endogenous expression level of β‐actin was quantified as an endogenous control with primer and probe sets for the nonhuman primate β‐actin (Overbergh et al., 2005 (link)) and the mouse β‐actin (Mouse ACTB Endogenous Control, 4352933E, Thermo Fisher Scientific). The levels of SARS‐CoV‐2 N gene were normalized to that of β‐actin. All qRT‐PCR assays were performed using the CFX96 Real‐Time PCR System (BioRad).

The HPAEpiCs were seeded at a density of 4×10⁵ cells/well in 24-well plates and grown overnight at 37 °C. Following preincubation with the test compounds for 2 hr, the cells were infected with SARS-CoV-2 at an MOI of 1. After 1 hr of incubation at 37 °C, the virus-drug mixtures were replaced with fresh medium containing compounds. After 24 hr, the cells were collected to extract total RNA and total cell protein. Viral RNA was quantified using a Thunderbird Probe One-step RT-qPCR Kit (QRZ-101, Toyobo, Shanghai, China). The TaqMan primers used for SARS-CoV-2 were 5’-GGG GAA CTT CTC CTG CTA GAA T-3’ and 5’-CAG ACA TTT TGC TCT CAA GCT G-3’ with SARS-CoV-2 probe FAM-TTG CTG CTG CTT GAC AGA TT-TAMRA-3’.

The number of viral genome copies in the Fcwf-4 cells infected with FCoV in each compound was determined using RT-qPCR [32 (link)]. Briefly, the medium was removed 20 h after infection, and RNA from the medium was prepared using Isogen-LS (Nippon Gene Co., Ltd., Toyama, Japan), according to the manufacturer’s protocol. Total RNA was reverse transcribed, and viral cDNAs were quantified using the THUNDERBIRD Probe One-step RT-qPCR kit (TOYOBO Co., Ltd., Tokyo, Japan) with specific primers for the gene encoding the FCoV nucleocapsid (GenBank: DQ010921.1; forward, 5′-TGGCCACACAGGGACAAC-3′; reverse, 5′-AGAACGACCACGTCTTTTGGAA-3′) and the TaqMan probe (FAM-TTCATCTCCCCAGTTGACG-BHQ-1). The sequences were amplified using a 7500 Sequence Detection System (Thermo Fisher Scientific, Tokyo, Japan).