Following is a scientist's responses to the question, "If an RNAi experiment results in insufficient gene knockdown, do you try the experiment again? If so, what approach do you use to improve the knockdown?"
For a complete list of scientists' responses, read the March issue of Genome Technology, a GenomeWeb News sister publication.
"In general we go through a triage process. Our first approach is to try the knockdown in another cell line that we know has high levels of RNAi activity. Second we tend to order a few more RNAi's around the target (provided we have not tried five or more in the first go around). If we have not seen transcript knockdown at this point, we may begin to suspect that the primer sets that we are using are incorrect, and so we will take another look at the same samples with new primers.
"When looking at protein levels we may suspect that antibodies are not very specific or that the protein has an unusually long half life. We may then use an epitope tagged cDNA overexpression approach to look for activity. When using this approach we prefer to overexpress the protein, wait 24 hours and then attempt the RNAi knockdown."
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Applied Genomics Bristol-Myers Squibb