Yes for the first.... No you Cannot for the second.. siRNA transfection almost leads to different percentage of knocking down (and not knocking out) depending on many factors, some of which : transfection efficiency, design of siRNA itself, gene type... many siRNA need to be tried and verified by western blot to see if more than 70% knockng down of the target gene has been occurred.
some proteins are still efficient even in minute amount please check if yours is not one these.. otherwise you can not use siRNA
@asmaa: well, and if i need my gene to get knocked down, and succeeded, how do i check out?
If not sirna, which techy will help me to do that?
I agree with Asmaa for the second point. It cant be "knocked out" it can just be "knocked down"
This meets my experience as well: You can knock it down, but not completely shut down. What gave me better results was the usage of shRNA plasmids which can be transfected into cells and then selected for. They are more expensive in the first hand, but if you use them a lot, it get cheaper, since you can grow them in bacteria yourself. And they are not as sensitive as naked (si)RNA is.
You can check success of knocking down by western blot..band of protein of interest should be lowered by more than 70%..I agree with @christian: For routine use of cells knocked down of your gene, use plasmids or viral transduction but I am not expert with that.
@Asmaa: Thats right. You should always check the protein expression, since downregulation in gene expression does not necessary mean that the protein is reduced a lot, too.
@christian: I am not much experienced with shrna. But I really need to check out after knocking down. Though I cant explain why I am doing ;), a multiple number of proteins need to be checked for. Do you think they might also be altered? (due to the reduction of the knocked down gene?)
@Gemitha: That depends on the protein you want to downregulate. If it is a signalling protein or a transcription factor it will most likely influence the expression of other genes as well. I did this for transcription factors and downstream genes where afffected. The first this to do was grow the cells with the shRNA plasmids, select and the prepare total RNA from them and look for gene expression. If this was down, we also did westerns to check for changes in protein expression. Does this help you, or do you have more specific questions?
Use lipofectamine/hiperfect for siRNA delivery. Try to buy silencing siRNA from good companies like CST. Check if particular gene knockdown has occurred by RTPCR and Western Blot
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