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 ...
