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stable siRNA - no downregulation after thawing (Jan/14/2005 )

I had transfected my C2C12 cells with specific siRNA. I got cells that survive the selection with hygromycin. At first I be able to detect a downregulation of the gene by using Real-Time-PCR. I have frozen these cells in liquid N2 for storage. When my cells died I thawed the cells and did a Real-Time-PCR but I could not detect a downregulation of the gene after thawing.

What do you think is the reason that the cells resistant to the antibiotic but do not express the siRNA?
Thanks a lot


do you mean you transfected with a shRNA expression plasmid? Assuming you do, it is likely that the shRNA your have expressed is not good against your target. A lot of shRNAs or siRNAs designed don't work against their targets. In the case of shRNAs, this is often the case, even if a corresponding chemically synthesised siRNA does work with the same sequence. This can be dues to several factors including (1) strange processing of teh shRNA into siRNA by dicer (2) The loop sequence of the shRNA being incorrect - different loop sequences work better with different shRNA sequences (3) assuming you have the order of your shRNA as 5'-sense - loop - antisense-3', knockdown can sometimes be improved by changing this to 5'-antisense-loop-sense-3'.

Hope this helps