siRNA knock down did not work - protein expression knock down by siRNA (Oct/16/2007 )

Hi,

I have used the Adenoviral Expression System to transduce siRNA to my target cells. On the siRNA vector, there is a co-expression marker CD25. 4-5 days after transduction, I measured both the expression of CD25 and the target protein by FACS.

I could see about 80-90% cells expressing CD25, however, nearly no knockdown of my target protein.

I have repeated this experiment for several times, and the knockdown of my target protein only woked once, which makes me think the problem may not be the siRNA. I also have a positive control for my target protein, and it works also very well. It means the problem may not be the staining either.

What hapened then?

Thank you for help!

Yanyan

-feilengcui1123-

Did you design the shRNA sequence yourself or get it from a paper? Perhaps it isn't quite right, or perhaps it got mutated during cloning (did you sequence to check for mutations?). Good luck

Dave

-miRNA man-

QUOTE (miRNA man @ Oct 16 2007, 10:03 PM)

Did you design the shRNA sequence yourself or get it from a paper? Perhaps it isn't quite right, or perhaps it got mutated during cloning (did you sequence to check for mutations?). Good luck

Dave

Thank you very much for answering!

Actually my supervisor has designed the shRNA, 4 different ones. Could I have so bad luck that all of them got mutated?

I will try another published shRNA for my target protein.

-feilengcui1123-

Sometimes one has to go through many siRNA sequences before getting the one with the best knockdown.

Good Luck !!!

-scolix-

Is it possible to use synthetic siRNA for that shRNA for a test transfection so that you can get a sense whether the siRNA works?

-pcrman-