ON-TARGETplus Human AR (367) siRNA - Individual

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Final concentration-10 nm. Add diluted siRNA to diluted Lipofectamine Reagent   Incubate for 5 min at RT and add to cells.  Replace medium after24h and re-incubate cells with 10 nM DHT or EtOH (vehicle) for 2 days at 37°C.

Protocol tips
Final concentration-10 nm. Add diluted siRNA to diluted Lipofectamine Reagent   Incubate for 5 min at RT and add to cells.  Replace medium after24h and re-incubate cells with 10 nM DHT or EtOH (vehicle) for 2 days at 37°C.

Publication protocol

To knock down endogenous CD9 expression, CD9-specific small interfering RNA (CD9 siRNA) was generated using published sequence ([80], 5′-GAGCATCTTCGAGCAAGAA-3′). Cells were seeded at 9 × 104 in six-well plates in RPMI + 5% FBS in RPMI 1640, and transfected with 5 nM CD9 siRNA after 72 hours with Lipofectamine RNAiMax (Invitrogen). Scrambled siRNA (CD9 scRNA; 5′-GGGAAUCGCCCAAAUAGAU-3′) was used as a negative control. Cellular proliferation was observed using IncuCyte HD system as described above 24 h after transfection. Forty-eight hours after transfection, cells were analyzed for knockdown efficiency by qRT PCR. To knock down endogenous AR expression, ON-TARGET plus AR siRNA (J-003400-07-0005, Thermo Scientific) was transfected on LNCaP grown in CSS for 24 h. Cells were treated with 10 nm AR siRNA or scRNA (D-001810-02) with Lipofectamine (Invitrogen) for 24 h, media were replaced and treated with 10 nM DHT or EtOH (vehicle) for 48 h.

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Manufacturer protocol

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