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A. If the RNAi sequence is getting into the cell and just not knocking it down enough, we will try another sequence or a combination of sequences. Invitrogen sells “smart pools” of RNAi’s consisting of four or five sequences. Sometimes you can get better knockdown just by shifting the sequence a couple of nucleotides. If the RNAi isn’t getting into the cell (test by uptake of a labeled oligo, e.g. Block it by Invitrogen) we will try other transfection reagents or may engineer into a viral vector (e.g. lentivirus, herpesvirus, etc.).

David Uhlinger
Senior scientist
Johnson & Johnson

 

A. At the moment, the best plan would be to make more shRNAs. There’s still no way to ensure that this will work, however.

William Hahn
Assistant Professor of Medicine
Harvard Medical School

 

A. 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.

Kevin FitzGerald
Group leader for emerging technologies, Applied Genomics
Bristol-Myers Squibb

 

A. Typically, insufficient knockdown in our hands results from some of these main factors (alone or in combination):

1. Stable protein which has a long half life will be hard to knock down in a timely manner. There is not much you can do about this other than plan the experiment to measure further time points.

2. Low transfection efficiency can also lead to overall low knockdown. Optimizing the transfection agent or method, cell growth rate, and other conditions for the transfection can usually help but some cell lines are difficult to transfect, limiting the extent of knockdown.

3. Not all siRNA target sequences are ideal, and in many cases, several need to be tested to find one that gives optimal results. In general, the success rate with siRNA designed with the most up-to-date target selection rules will be higher than 50 percent, but this is very gene sequence-dependent and there may be limits to the achievable knockdown in certain genes where there simply are no good target sequences.

4. When all else fails, it may be the purity of the siRNA, or its integrity. We favor high-quality synthesis of siRNA from reputable vendors, and we avoid pooling our siRNA to avoid problems associated with off-target effects of bad siRNA in a mix, and to avoid needing to de-convolute a mixture to figure out which siRNA target sequence worked best. The storage of the siRNA and handling may also lead to degradation of the siRNA, and sometimes the simplest solution is to use control siRNA and reorder siRNA that appear to be degraded.

If an siRNA is not giving the desired knockdown, one should avoid increasing the concentration too high as this may lead to non-specific effects.

Spyro Mousses
Director, Cancer Drug Development Laboratory
Translational Genomics Research Institute

 

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