Power sybr green pcr 2 x master mix

MDCK cells were grown to about 90% confluence, infected with influenza virus at 0.001 MOI and cultured in the presence of 18-hydroxyferruginol (1), 18-oxoferruginol (2), and oseltamivir. The infected/untreated cells were cultured in the presence of 0.5% DMSO. The medium was removed after 3, 6, 12, and 18 h. Cells were scraped off, washed twice with phosphate buffer saline (PBS), and collected by centrifugation (500× g for 3 min). The total RNA was isolated using the Qiagen RNeasy mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The specific primers were used for the reverse transcription of viral RNA (vRNA; M gene, 5′-CTTCTAACCGAGGTCGAAACGTA-3′ and 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; NP gene, 5′-AGRTAYTGGGCYATAAGRAC-3′ and 5′-CTTATTTCTTCGGAGACAATGC-3′). GAPDH was used as the internal control for cellular RNAs, with primer sequences of 5′-TCAACGGATTTGGCCGTATTGG-3′ and 5′-TGAAGGGGTCATTGATGGCG-3′. cDNA was synthesized from total RNA using the cDNA Master Mix (Applied Biosystems, CA, USA). qRT-PCR was conducted using 2 μL of cDNA and the Power SYBR Green PCR 2 X Master Mix (Applied Biosystems, CA, USA). qRT-PCR was conducted using the Step One Plus Real-time PCR System, and the data were analyzed with the StepOne software v2.1 (Applied Biosystems, CA, USA).