ON-TARGETplus Mouse Ddit4 (74747) siRNA - SMARTpool

Protocol tips

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	The culture medium of RGC-5 cells (seeded at 5 × 104 cells/ml) was changed after 16 h with transfection medium. The transfection medium lacked serum and antibiotics and consisted of Lipofectamine RNAiMAX with 50 nM siRNA (dissolved in OPTIMEM medium) or without siRNA (mock). After 4 h, the transfection medium was removed and replaced with culture medium lacking antibiotics. After an additional 6 h, the cells were used for various studies when the down-regulation of mRNA was approximately 70–80%. To apply siRNA, ON-TARGET plus SMART pool, mouse DDIT4 or RTP801 (Thermo Scientific Dharmacon), a pool of four different target sequences were used. Controls routinely were mock transfections rather than ON-TARGET plus Non-targeting pool transfections, as they gave similar results.

Protocol tips

The culture medium of RGC-5 cells (seeded at 5 × 104 cells/ml) was changed after 16 h with transfection medium. The transfection medium lacked serum and antibiotics and consisted of Lipofectamine RNAiMAX with 50 nM siRNA (dissolved in OPTIMEM medium) or without siRNA (mock). After 4 h, the transfection medium was removed and replaced with culture medium lacking antibiotics. After an additional 6 h, the cells were used for various studies when the down-regulation of mRNA was approximately 70–80%. To apply siRNA, ON-TARGET plus SMART pool, mouse DDIT4 or RTP801 (Thermo Scientific Dharmacon), a pool of four different target sequences were used. Controls routinely were mock transfections rather than ON-TARGET plus Non-targeting pool transfections, as they gave similar results.

Publication protocol

The culture medium of RGC-5 cells (seeded at 5 × 104 cells/ml) was changed after 16 h with transfection medium. The transfection medium lacked serum and antibiotics and consisted of Lipofectamine RNAiMAX with 50 nM siRNA (dissolved in OPTIMEM medium) or without siRNA (mock). After 4 h, the transfection medium was removed and replaced with culture medium lacking antibiotics. After an additional 6 h, the cells were used for various studies when the down-regulation of mRNA was approximately 70–80%. To apply siRNA, ON-TARGET plus SMART pool, mouse DDIT4 or RTP801 (Thermo Scientific Dharmacon), a pool of four different target sequences were used. Controls routinely were mock transfections rather than ON-TARGET plus Non-targeting pool transfections, as they gave similar results.

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