I am knocking down each member of a family of 3 proteins, however I'm noticing that two members are getting knocked down at once due to high sequence homology (80% with BLAST). Is there a way I can reduce this effect to get a specific knockdown?
Thanks for reading :)
It will be difficult to eliminate the effect associated with 80% protein (gene?) homology by changing the conditions of WB binding. Try using another siRNA that covers a non-homologous region (within 20%) of your target that needs to be silenced.
As pointed by Sakanyan, use another siRNA that targets non-homologous region. You may find the siRNA from Thermo Scientific (Silencer Select Pre-Designed & Validated(Ambion)). They generally provide three siRNA that aim at different sites. You will get details of the siRNA and its target region from its description.
In order to prevent the binding to other targets, you need to the increase specificity of your siRNA, therefore find a sequence that is significantly different from any on the homologous genes (non-homologous regions).
The position inside the gene does not matter.
You can check the specificity by aligning your candidate sequence against the genome of the host, not just the homologous genes.
You can also play on the sequence length:
A shorter sequence has an higher probability to have more matches, so less specific.
A longer sequence is has a lower probability to have more matches, so more specific.
The method you use fixes a length range, because a minimum length is required to allow the binding, but there is also a maximum length, that could make the construct unstable. For instance VIGS requires a minimum of 20 nt for a good match and the longer it is the less stable and more transient is the expression.
Unlike primers, you cannot change the conditions to facilitate the binding.
Thermo Fisher provides a service for developing siRNA on demand.
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