Dusp3 siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-50 nM Incubate cells for another 24h at 37°C.

Protocol tips
siRNA concentration-50 nM Incubate cells for another 24h at 37°C.

Publication protocol

To test the role of each candidate gene on cytokine production by host defense cells, we transfected siRNAs into the mouse macrophages. All siRNAs were purchased from Invitrogen. RAW264.7 cells or bone-marrow derived macrophages from C57BL/6J were transfected with 50 nM siRNA by Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Twenty-four hours post-transfection, cells were treated with S. aureus Bioparticles (Invitrogen) to a final concentration of 10 µg/ml. At 24 hours post-infection, supernatants were collected and stored at −80°C for Luminex-multiplex cytokine assay. In parallel experiments, cells at 3 hours post-infection were harvested for RNA extraction by RNeasy (QIAGEN), RT-PCR by SuperScript II (Invitrogen) and SYBR Green qPCR analysis (ABI) respectively. Experiments were repeated at least three times. A full list of gene names and siRNA ID numbers were in Table S1.

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Manufacturer protocol

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