Triple up and rescue

About the image: Biotin-labeled siRNA (brown) in mouse lung tissue. Credit: Courtesy of Catherine Taylor" />About the image: Biotin-labeled siRNA (brown) in mouse lung tissue. Credit: Courtesy of Catherine Taylor User: Catherine Taylor, University of Waterloo, Canada Project: Using siRNA knockdown to identify proteins involved in apoptosis Problem: As in Ruiz-Vela's case above, SiRNAs can induce off-target effects. Controls: Tayl

Melissa Lee Phillips
Feb 1, 2008
<figcaption>About the image: Biotin-labeled siRNA (brown) in mouse lung tissue. Credit: Courtesy of Catherine Taylor</figcaption>
About the image: Biotin-labeled siRNA (brown) in mouse lung tissue. Credit: Courtesy of Catherine Taylor

User:
Catherine Taylor, University of Waterloo, Canada

Project:
Using siRNA knockdown to identify proteins involved in apoptosis

Problem:
As in Ruiz-Vela's case above, SiRNAs can induce off-target effects.

Controls:
Taylor always performs separate experiments with different siRNA sequences - each designed to knock down the same mRNA, but by targeting a different part of the transcript. It's "absolutely crucial," says Taylor. "I never do any experiments without it." She learned her lesson when she once used just one siRNA to suppress a gene and obtained a result she was expecting from previous work. "I was convinced it was correct, and I didn't include the second siRNA right away. When I did, I found it didn't behave the same as the first siRNA," she says. "Sure enough, it was due to some other effect."

Taylor also uses one of these triplets for a rescue experiment - the ultimate siRNA control. She designs one of the positive controls to target the 3' UTR (untranslated region) in her gene of interest. After knocking down target gene expression with the siRNA, she then transfects her cells with a form of the gene missing the UTR. If that version, which is insensitive to RNAi, restores normal function, then she knows its decreased expression was caused by the altered phenotype.

"Rescue experiments can be inexpensive if you have an RNAi-resistant cDNA already available to you, and if your cell line is easy to transfect," Taylor says. The price increases, however, if you need to mutate your gene of interest to make it RNAi-resistant or develop a viral-expression vector to increase transfection efficiency.

Cost:
$350-$400 per siRNA from Dharmacon; $250 for 0.75 mL Lipofectamine 2000 transfection reagent from Invitrogen

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