hi everyone,
I am recently performing RNAi rescue assay to validate the siRNA for gene/protein A specificity. I construct cell lines expressing wild type gene/protein A and RNAi resistant gene/protein A (that is, the mRNA sequence corresponding to siRNA were mutated to another condon, but coding the same amino acid, so that RNAi will not interefere the RNAi resistant gene/protein A expression). Unfortunately, cells expreesing resistant gene/protein A cannot rescue the phenotype caused by siRNA for gene/protein A. Noteworthy, RNAi resistant gene/protein A indeed cannot be interefered by siRNAs. And the phenotype we observed had been reported by another two groups previously. More important, different siRNAs sequence were used by us and the other two group. So, does anyone know the reason for such weird thing?
Thanks in advance!
Zhiyuan
Have you verified that the suposedly resistant gene is not being knocked down (using e.g. RT-qPCR)?
The only thing I can think of is that your RNAi-resistant gene isn't being expressed correctly. Have you verified that it is expressed?
Also, as a curiosity, I don't understand your "gene/proteinA" nomenclature. Are you expressing a fusion of your protein and protein A?
hi Alejandro, we just verified the knockdown efficiency for the suposedly resistant gene by Western blot. And it showed not being knocked down. ( as the attachment indicated)
Hi Michael,
We have verified the expression for RNAi-resistant gene. (as indicated response to Alejandro in above). Do you mean that the RNAi-resistant protein in non-functional?
Gene/protein A just mean the protein A or Gene A alone, not a fusion protein. Sorry about this.
Thanks Amit. But I just cannot rescue the RNAi effect in RNAi-resistant gene expressing cells.
Taking account of all of that, it is possible that you are getting some off-target effects of your siRNA - i.e., it is knocking down another gene which is resulting in your phenotype.
Do you find any sequence similarity with any other genes?
Either that or, as you say, your RNAi-resistant protein is non-functional.
According to the Blast results, the siRNA sequence did not show any similarity. Given the fact that the phenotype induced by the same gene had been reported by other group, it is possible that RNAi-resistant protein in non-functional.
Thanks, Michale.
Out of curiosity, do you have a hypothesis as to why the RNAi-resistant protein would be non-functional? After all, it does have the same aminoacid sequence as the RNAi-sensitive variant, right?
Hi Alejandro,
There was a paper indicating your curiosity.
see the attachment.
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