nucleic acid extraction from silenced cells - (Jul/15/2010 )

Hi all!
I want to perfom gene silencing on cells seeded in a 24-well plate. I would like to control by semi-quantitative pcr the expression of the transcript of interest, but I'm afraid that cells in a well are not enough to perform acid nucleic extraction. Can I trypsinize cells and allow them to multiply for a day in a T25 flask, for example 12 hours after transfection with siRNA?

Hope can you give me suggestions!
Thank you.

AnnaSh on Jul 15 2010, 12:57 PM said:

Sure you can do it.

Do you have any selection marker you can use to select the silenced cells?

Thanks for replying,
I have not a reporter gene to select silenced cells. The kit I use just suggests to monitor the expression of my target by rt-pcr or western blot analysis on the whole cultures. Do you think it is not a perfect procedure?
I have also another doubt. My target protein is involved in a specific physiological mechanism (that I want to study functionally), that also involves other proteins. I am right if I monitor the expression of these proteins in silenced cells, in the attempt to depict a cell regulatory strategy? I'm afraid that being silencing a "forced" action, the regulatory events I can observe are not real...is it true?

Thank you again for your kindness.

AnnaSh on Sat Jul 17 14:25:25 2010 said:

If you detect the protein by WB and you can see the inhibition it is a perfect control.
Well, if you "remove" a protein from the cell you will have a result...the point is that you will have some effect in some pathway (not in all of them), so you can say that your protein is involved in that pathway; Obviously you will have to do other experiments to prove your theory, but showing how the silencing of your protein affects some given pathway is a good approach.

Thanks for your reply!
I just want to submit an example to you. Suppose I silenced my target protein. Obviously I can study short-term phenomenona mediated by this protein, like cell transport... and that's ok. Imagine I found an increased/decreased expression of other proteins. Can I say that my target regulates their gene expression? I've some doubts about, because the mechanism of silencing consists of the disruption of mRNA, that is not physiological. In other words, I don't interfere with a physiological pathway to achieve silencing, so I'm afraid that results are not real...

Thanks in advance.

AnnaSh on Tue Jul 27 08:21:24 2010 said:

well, first you should check if that increased/decreased that you see in other proteins is specific. If you are working with a TF (activator or repressor) it would be perfectly normal that your protein affect the transcription of some target genes. Anyway sometimes you can affect somehow the stability of other proteins (by direct interaction,sequestering, phosphorylation, sumoylation, transport...). And if that is due to the fact that your protein is not there (and not by the silencing itself)then I would say it is a result. And the "silencing" itself could not be physiological, but the result could mimic a physiological scenario. And it is a perfectly good approach to check the function of your protein. The same point could be made against knock out cells (because it is not a physiological scenario), but they are perfectly fine to find out if your protein is involved in a given process.

Now I have a clearer idea about the information I can get from silencing experiments.

Thanks for sharing your expertise!