We are knocking down TIGIT mRNA with siRNA and see near complete reduction of the mRNA but only slight to moderate reduction of the surface protein. We have looked at longer time points and multiple rounds of transfection with no additional reduction in surface proetin seen. Thanks!
Hi Melissa,
To preface, I am not familiar with the target gene nor your cell line but perhaps I can give you some insight. I would ask and see if your controls for this experiment are working or not, especially the positive control. If your lab has never successfully accomplished this particular knockdown in this particular cell line, then you have a lot of ground to cover.
How was the siRNA designed? Can you design two more siRNA sequences to test? Criteria that would optimize siRNA knockdown include: 1. sequence location (should be within 50-100 nt of the start codon or termination codon to ensure gene silencing), 2. GC content (ideally 40-50%), and 3. off-target events (I recommend a BLAST search to prevent off targeting).
I would also investigate with a dose response curve, to see if knockdown takes place at a higher siRNA concentration (from which you can then optimize through this study). A study based on time degradation would be useful, to assess the amount of time needed for knockdown to take effect (in general, larger molecules have long half lives).
Hello, Melissa.
I am studying the NK cells as well. Previously I use lipo 3000 and lipo RNAiMAX to transfect the siRNA into NK cell. However, it seems useless. The siRNA almost cannot enter into NK cell. So I want to ask how you transfect the siRNA.
Chunhao Lin , we have self-delivering RNAi compounds that we are using.
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