Recombinant taq polymerase

RAW 264.7 cells (5 × 10⁵ cells/60 mm dish) were stimulated for 6 h with or without test compounds in the presence of LPS (1 μg/mL). Total RNA was extracted by TRIzol (Life technologies, Frederick, MD, USA) according to the manufacturer’s instructions and then reverse transcribed into cDNA using reverse transcriptase (Life Technologies, Frederick, MD, USA) and random hexamer (Cosmo, Seoul, Korea). Then, prepared aliquots of cDNA were amplified using a recombinant Taq polymerase (Promega, Madison, WI, USA) and primers for iNOS and β-actin to detect gene expression of iNOS and β-actin.

Cells (1.8 × 10⁶ cells/60mm dish) were stimulated for 6 h with LPS (1 μg/mL) in the presence or absence of test compounds. Total RNA was extracted by TRIzol (Life technologies, Frederick, MD, USA) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using reverse-transcriptase (Life technologies, Frederick, MD, USA) and random-hexamer (Cosmo, Seoul, Korea). The cDNA amplification was performed by using a recombinant Taq polymerase (Promega, Madison, WI, USA) and primers for iNOS, COX-2, IL-6, IL-1β, TNF-α, and β-actin. The amplified PCR products were separated on 2% agarose gels and stained with ethidium bromide.