ON-TARGETplus Human THBS2 siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 5,000 cells/well in a 96-well plate	siRNA concentration-0.25μg–1.0μg. Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate cells for 1–3 days at 37°C.

Upstream tips
Seed 5,000 cells/well in a 96-well plate
Protocol tips
siRNA concentration-0.25μg–1.0μg. Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate cells for 1–3 days at 37°C.

Publication protocol

siRNAs were obtained from Dharmacon (Lafayette, CO) as follows: 1) non-targeting-non-fluorescent control siRNA (D-001206-13-20), 2) siGLO Red®, a transfection indicator, consisting of non-targeting double stranded siRNA conjugated to the red fluorescent probe DyLight547, 3) 3′ Cholesterol and a 5′ prime Dy547 tagged non-targeting control siRNA (Chol siRNA), 4) TSP-2 siRNA and 5) 3′ Cholesterol TSP-2 siRNA (Chol TSP-2 siRNA). Two transfection reagents were procured: 1) Lipofectamine RNAiMAX® (liposomal transfection reagent; Invitrogen, Carlsbad, CA) and 2) jetPEI™ (a cationic polymer; Polyplus, Strasbourg, France).

Primary human aortic smooth muscle cells (AoSMC) (Lonza, Walkersville, MD) were cultured in basal medium (LifeLine, Walkersville, MD) enriched with SMC growth supplements. The cells were maintained at 37°C with 5% CO2. AoSMCs from passages 4–8 were used in the experiments. AoSMC were seeded at a density of 5,000 cells/well in a 96-well plate (Fisher Scientific, Pittsburgh, PA). After 24 hours, AoSMCs were transfected with conditions described in Table 1. For all transfections, siRNA was used in the range of 0.25μg–1.0μg. All transfection reagents were used as recommended by the manufacturer. Complexation of siGLO Red or Chol siRNA with PEI at N/P ratios of 2.5, 5 and 10 were evaluated for delivery efficacy (N/P ratio = ratio of moles of amine groups of PEI to moles of siRNA phosphate groups).

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Manufacturer protocol

Download the product protocol from Dharmacon for ON-TARGETplus Human THBS2 siRNA below.

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