PACE4 siRNA (h)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	PACE4 siRNA concentration-100 nM Add incubated siRNA mixture (5 minutes at RT) and transfection mix (5 minutes at RT) to cells. Incubate at 37°C for 24 to 72 hours.	In case of Low Transfection Efficiency use buffer (100mM NaCl, 50mM Tris pH7.5 in RNase-free water) to dilute siRNA; water only denatures siRNA to improve the siRNA quality. In case of cell cytotoxicity, use less GeneSilencer(GS) Reagent and also lower the amount of siRNA used to keep GS:siRNA ratio optimized

Protocol tips
PACE4 siRNA concentration-100 nM Add incubated siRNA mixture (5 minutes at RT) and transfection mix (5 minutes at RT) to cells. Incubate at 37°C for 24 to 72 hours.
Downstream tips
In case of Low Transfection Efficiency use buffer (100mM NaCl, 50mM Tris pH7.5 in RNase-free water) to dilute siRNA; water only denatures siRNA to improve the siRNA quality. In case of cell cytotoxicity, use less GeneSilencer(GS) Reagent and also lower the amount of siRNA used to keep GS:siRNA ratio optimized

Publication protocol

hree human PCa cell lines, DU145, LNCaP, and PC3 cells, were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were routinely grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone®; Thermo Fisher Scientific Inc, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) (Hyclone; Thermo Fisher Scientific Inc), 100 U/mL penicillin (Sigma-Aldrich Corp, St Louis, MO, USA), and 100 μg/mL streptomycin (Sigma-Aldrich Corp) in a humidified atmosphere of 5% CO2 at 37°C.

Cells were transfected with 100 nM PACE4 siRNA (Genbank ID for PACE4: NM_001291309) (sc-43990; Santa Cruz Biotechnology Inc, Dallas, TX, USA) or control siRNA (scrambled siRNA, a universal negative control), using GeneSilencer siRNA Transfection Reagent (Genlantis, San Diego, CA, USA), according to the manufacturer’s instructions. At 48 hours after transfection, the efficiency of siRNA-mediated PACE4 knockdown was determined by western blot.

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Manufacturer protocol

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