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.
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.
Director, Cancer Drug Development Laboratory
Translational Genomics Research Institute