RNA was extracted using TRIzol reagent (ThermoFisher Biochemical Products, Beijing, China). The cDNA was synthesized using a Prime Script RT reagent kit (TaKaRa Bio Inc, Dalian, China) and then used as the template for real-time qPCR using the TB Green Premix Ex Taq mix kit (TaKaRa Bio Inc., Dalian, China). RT-qPCR assays were performed on a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Singapore) and the software used was Bio-Rad CFX Manager 3.1(Bio-Rad Laboratories, Singapore) for 40 cycles of denaturation (96 °C for 5 s), reannealing, and extension (60 °C for 30 s). The relative mRNA level of these genes was normalized to the porcine GAPDH mRNA level. mRNA was calculated and normalized based on the comparative cycle threshold (2−ΔΔCt) method [40 (link)]. The primers of each gene are shown in Table 2 .
Tb green premix ex taq mix kit
Manufactured by Takara Bio
Sourced in China, Japan
The TB Green Premix Ex Taq mix kit is a real-time PCR reagent solution that contains TB Green dye for the detection and quantification of DNA targets. The kit includes a Premix solution with DNA polymerase, dNTPs, and TB Green dye.
Automatically generated - may contain errors
2 protocols using tb green premix ex taq mix kit
1
RNA Extraction and RT-qPCR Analysis
2
Quantification of ASFV RNA Expression
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BMDM cells were infected with 0.1 MOI of ASFV CN/GS/2018 or ASFV-Δ2R in six-well plates. For indicated durations, whole cellar RNA was extracted with TRIzol reagent (Invitrogen, USA; Cat# 15,596,026) and subjected to reverse transcription using Prime Script RT reagent kit (Takara, Japan; Cat# RR037A) according to the manufacturer's instructions. The resulting cDNA was then used as the template for real-time qPCR using the TB Green Premix Ex Taq mix kit (Takara, Japan; Cat# RR402B). The results were depicted by the 2−ΔΔCt method and expressed relative to the control sample (Schmittgen and Livak, 2008 (link)). All the RT-qPCR primers used were listed in Supplementary Table S2 .
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