mRNA versus protein

About the image: Neuro2A cells transfected with a control siRNA (A) or a siRNA against the protein AP-3Δ (B), and stained 48 hours later with an AP-3Δ antibody (green). The transfection leads to a marked decrease in AP-3Δ levels. Credit: Courtesy of Raphael Rozenfeld" />About the image: Neuro2A cells transfected with a control siRNA (A) or a siRNA against the protein AP-3Δ (B), and stained 48 hours later with an AP-3Δ antibody (green). The transfection leads to

Melissa Lee Phillips
Feb 1, 2008
<figcaption>About the image: Neuro2A cells transfected with a control siRNA (A) or a siRNA against the protein AP-3Δ (B), and stained 48 hours later with an AP-3Δ antibody (green). The transfection leads to a marked decrease in AP-3Δ levels. Credit: Courtesy of Raphael Rozenfeld</figcaption>
About the image: Neuro2A cells transfected with a control siRNA (A) or a siRNA against the protein AP-3Δ (B), and stained 48 hours later with an AP-3Δ antibody (green). The transfection leads to a marked decrease in AP-3Δ levels. Credit: Courtesy of Raphael Rozenfeld

User:
Raphael Rozenfeld, Mount Sinai School of Medicine, New York

Project:
Using siRNA knockdown to identify proteins involved in opioid and cannabinoid receptor signaling

Problem:
siRNA might depress mRNA levels but not induce the expected phenotype.

Controls:
Looking for a decrease in levels of your targeted mRNA isn't enough, Rozenfeld says; you should also look for reduced levels of that mRNA's protein. "Make sure that your siRNA leads to a specific decrease in the targeted protein," he says - otherwise, you won't see a phenotype. You'll need to know your protein's half-life to estimate the timeframe in which mRNA knockdown should affect your protein levels.

"Although it is often recommended testing whether the siRNA worked 48 to 72 hours after the knockdown, it is also best to check it after 24 and 96 hours," suggests Pekovic, to ensure that you don't miss the effects of particularly short- or long-lived proteins.

Rozenfeld also assays levels of proteins from the same family as his target protein, to provide additional proof that only the target protein is downregulated.

Cost:
$300 per siRNA from Dharmacon and Santa Cruz Biotechnology; $250 for 1 mL Lipofectamine transfection reagent from Invitrogen

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