siRNA: how long will knock-down last - (Nov/29/2006 )

hi guys,

i am going to start some RNA interference work soon and wonder how long after i transiently transfect my cells with siRNA will the gene of interest remain knocked down. most propreitry kits state to asses knockdown after 72 hours, but does that mean the gene won't be knocked down for much longer after that? also, can i passage my cells after they've been transfected, they grow enormously fast

regards

well, siRNA transfected are supposed to retain in proper cell concentration till 96h as i saw in publications routinely.
If you want longer silencing, you should use expression vectors.
Btw, i'm now facing cell adaptation to siRNA in my experiment after 9weeks of silencing an essential protein.

adapting as in the siRNA is no longer knocking down the gene???

well it was a siRNA against a essential protein.
the clone took 9 month to be visible on a petri dish
now the target protein is not down regulated as in the siRNA transfection assay,and moreover, the cell ine growth like a normal one. not done alread the siRNA expression analysis, but seems the clone that have survived to that is one that have partially silenced the siRNA expression or sthg like that

Hi Fred_33

may i ask which method you use to check if the shRNA (i assume that is what you mean by stable clone) is really expressed? Commercial kit? Northern?

Stardust

ok the day have well started...

I'm not able to see the siRNA by northern but it's the most used wqy to do that.
if youwant to use a commercial kit, use mirVana from ambion

so your clone does not express the shRNA?

Is it a random integration in the genome of the cell? How many copies do you have integrated? What kind of probes are you using for northern? LNA or DNA or RNA probes? What type of labelling?

Stardust