negative control siRNA - (Sep/01/2005 )

Theo22 is right, rescue is a good control. The point is that's not always possible to do it. For instance I transfect siRNA in BAEC which is working great whereas dsDNA do not transfect. I must infect and select them to express the mutated protein insensitive to siRNA but that's not easy as BAEC are primary cells and I cannot keep them in culture for long.

However, this is working well in HeLa cells or this kind of cells and you can even express mutant forms of your protein if you want to assess a specific question such as if any enzymatic activity is necessary for the phenotype...

Yes, I realise that some cell-types are very difficult to work with.....
Sometimes this makes it difficult to draw conclusions from functional studies iperformed in those cells.

Well, don't you think that finally the best is to use several different controls (several siRNAs, several negative controls, rescue...).

I thought about it because people are discovering new things on siRNA everydays and controls that may look not necessary today may be crucial tomorrow. For instance, I've been working with siRNA in human cells since the discovery of silencing in mammalian cells but at the beginning studies (including ours) were using a single siRNA what I wouldn't trust at all now.

I realize that's maybe too much if you just want to check if a given protein is involved in the process you're studying but if you try to perform screenning processes with libraries or this kind of things, I'm always afraid of artifacts and off-target effects.

Thanks for the discussion Theo22, very informative and I need to be stimulated!!