Luna universal one step rt qpcr kit

Huh7 cells were seeded at a density of 50,000 per well in a 48-well plate. The next day, the cells were infected with either DENV-2 at an MOI of 0.01 CCID₅₀/cell, purified DENV DIPs (equivalent to 1,000 DI-290 RNA copies per cell), or DENV-2 mixed with purified DENV DIPs. For poly(I:C) stimulation, the cells were treated with poly(I:C) (Sigma-Aldrich) at a concentration of 5 μg/mL. Total RNA from cells was extracted after 2, 24, and 72 h postinfection using an RNeasy kit (Qiagen), and the mRNA expression levels of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), IFN-α, IFN-β, OAS1, PKR, and ISG15 were determined using a Luna Universal One-Step RT-qPCR kit according to the manufacturers’ instructions. The primer sequences are available upon request. The data were normalized to RPL13a as the reference gene and presented as fold change relative to untreated, uninfected control cells using the threshold cycle (ΔΔCT) method.

Huh7 cells were seeded at a density of 50,000 per well in a 48-well plate. The next day, the cells were infected with either DENV-2 at an MOI of 0.01 CCID₅₀/cell, purified DENV DIPs (equivalent to 1,000 DI-290 RNA copies per cell), or DENV-2 mixed with purified DENV DIPs. For poly(I:C) stimulation, the cells were treated with poly(I:C) (Sigma-Aldrich) at a concentration of 5 μg/mL. Total RNA from cells was extracted after 2, 24, and 72 h postinfection using an RNeasy kit (Qiagen), and the mRNA expression levels of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), IFN-α, IFN-β, OAS1, PKR, and ISG15 were determined using a Luna Universal One-Step RT-qPCR kit according to the manufacturers’ instructions. The primer sequences are available upon request. The data were normalized to RPL13a as the reference gene and presented as fold change relative to untreated, uninfected control cells using the threshold cycle (ΔΔCT) method.