Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells

Law, Helen KW; Cheung, Chung Yan; Sia, Sin Fun; Chan, Yuk On; Peiris, JS Malik; Lau, Yu Lung

Total RNA was extracted from ~1.5 × 10 5 cells harvested at 3 h and 9 h post infection by TRIzol Reagent (Invitrogen Life Technologies, USA). In later experiments, QiaShredder columns (Qiagen, Hilden, Germany) were used to ensure adequate homogenisation and RNA was extracted by the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed on the DNasetreated total RNA using oligo (dT) primers and Superscript II reverse transcriptase (Invitrogen Life Technologies, USA) according to the manufacturer's recommendation. The cDNA synthesised were diluted (1:50) and quantified by real-time PCR using Taqman Technology (Applied Biosystems, CA, USA). Specific primers ( Table 2) were used and non-specific reactions and primer-dimer artifacts have been minimised (as evaluated by gel electrophoresis). Detection of PCR product was based on Taqman fluorescence signal. Standard curves were generated using serial dilutions of plasmids (~10 -10 10 copies) containing cloned sequences involved. Results were calculated as the number of targeted molecules/μL cDNA. To standardise results for variability in RNA and cDNA quantity and quality, we express the results as the number of target copies per 10 4 copies of β actin gene, which was determined previously [8] .