Human gapdh primer probe mix

Huh-7 cells were infected with DENV and at indicated time points, cells were collected in TRIzoL reagent (Takara) and RNA was isolated using manufacturer’s instructions. cDNA synthesis was performed using PrimeScript RT reagent kit with gDNA eraser (Takara). 100 ng of cDNA was used to determine genes expression using DyNAmo flash SYBR green quantitative PCR reagent (Thermo Scientific). Reaction conditions used were as follows: (95°C-7 min; 95°C-10 s followed by 60°C for 30 s). GAPDH primer was used as housekeeping control. For DENV RNA detection by reverse-transcription polymerase chain reaction (RT-PCR), total RNA was extracted from cells at the indicated time points using RNAiso Plus (TaKaRa), and 200 ng of RNA was used in multiplex TaqMan one-step RT-PCR with DENV primers, DENV probe and human GAPDH primer-probe mix (Applied Biosystems). At indicated time points, supernatant was collected for estimating viral titers by plaque assay and cells were harvested for positive and negative strand detection PCR as described previously (Kar et al., 2017 (link)). Expression levels of GAPDH was used to calculate fold change and normalisation. Data were analysed using the ΔΔ C_T method, where C_T is threshold cycle.