14-3-3ζ siRNA(h)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-5 μmol/L Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Incubate cells for 2 days at 37°C.

Protocol tips
siRNA concentration-5 μmol/L Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Incubate cells for 2 days at 37°C.

Publication protocol

The small interfering (si)RNA that was designed to specifically target the coding region of HuR (siHuR) or 14‐3‐3ζ (si14‐3‐3ζ) mRNA were purchased from Santa Cruz Biotechnology. Scrambled control siRNA (C‐siRNA), which had no sequence homology to any known genes, was used as the control. The siHuR, si14‐3‐3ζ, or C‐siRNA was transfected into cells as described previously (Rao et al. 2002, 2008, 2010; Chung et al. 2015). Briefly, for each 60‐mm cell culture dish, 20 μL of the 5 μmol/L stock siHuR, si14‐3‐3ζ, or C‐siRNA was mixed with 500 μL of Opti‐MEM medium (Invitrogen). This mixture was added to a solution containing LipofectAMINE 2000 in 500 μL of Opti‐MEM. The solution was incubated for 20 min at room temperature and gently overlaid onto monolayers of cells in 3 mL of medium, and cells were harvested for various assays after 48 h incubation.

The chimeric firefly luciferase reporter constructs containing different fragments of 14‐3‐3ζ mRNA were generated by the procedure described previously (Wang et al. 2010; Liu et al. 2015). The full‐length 14‐3‐3ζ 5′ UTR, CR, or different fragments of 3′ UTR were amplified and subcloned into the pmirGLO‐Luciferase miRNA Target Expression Vector (Promega, Madison, WI) to generate the pmirGLO‐LUC‐14‐3‐3ζ – 5′ UTR, pmirGLO‐LUC‐14‐3‐3ζ – CR, and pmirGLO‐LUC‐14‐3‐3ζ – 3′ UTR (F1–F3). The sequence and orientation of the fragment in the luciferase reporter were confirmed by DNA sequencing and enzyme digestion.

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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for 14-3-3ζ siRNA(h) below.

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