Order in the house

About the image: The nuclear protein LAP2alpha (left panels) expresses a cell proliferation marker (K167, middle panels) when measured with a positive RNAi control (top row) but not upon LAP2a siRNA knockdown (bottom row). Right panels: merged images with DAPI cellular stain. Credit: Courtesy of Vanja Pekovic" />About the image: The nuclear protein LAP2alpha (left panels) expresses a cell proliferation marker (K167, middle panels) when measured with a positive RNAi control (top row) but not upon

Melissa Lee Phillips
Feb 1, 2008
<figcaption>About the image: The nuclear protein LAP2alpha (left panels) expresses a cell proliferation marker (K167, middle panels) when measured with a positive RNAi control (top row) but not upon LAP2a siRNA knockdown (bottom row). Right panels: merged images with DAPI cellular stain. Credit: Courtesy of Vanja Pekovic</figcaption>
About the image: The nuclear protein LAP2alpha (left panels) expresses a cell proliferation marker (K167, middle panels) when measured with a positive RNAi control (top row) but not upon LAP2a siRNA knockdown (bottom row). Right panels: merged images with DAPI cellular stain. Credit: Courtesy of Vanja Pekovic

User:
Vanja Pekovic, University of Durham, UK

Project:
Using siRNA knockdown to establish a protein's role in fibroblast proliferation and cell-cycle regulation

Problem:
When an siRNA doesn't knock down the expression of its target, the problem isn't always clear.

Controls:
Test your set-up with an siRNA whose effects you know in advance. Pekovic runs her positive controls with an siRNA that targets the human lamin A/C gene, but lamin B also works well, she says. "These proteins are so abundant and give very specific staining in the nucleus, [so they] can be easily assessed for the effectiveness of the siRNA method."

She ordered three versions of this siRNA from Ambion, but not all of them worked well in her human cell lines. The success of each siRNA "most likely depends on the cell type and conditions used, which is why they have three different ones," she says. The only way to be sure of which siRNA will work best in your cells is to try them all. Some people prefer to use all three at once, at low concentrations, Pekovic adds.

Other common positive controls are siRNAs against HPRT (hypoxanthine-guanine phosphoribosyl transferase) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase), according to Behlke. "If you don't see 95% knockdown with your positive control, then you're not going to be able to get good results with your gene of interest."

Cost:
$500 for three siRNAs from Ambion; $250 for 1 mL Oligofectamine transfection reagent from Invitrogen.

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