Does Lipofectamine cause gene knock down?
Sudheendra Rao N R
(by sudhee26 from gmail.com)
Thu May 8 10:14:05 EST 2008
quite scary i must say :)
but if i am getting u right, u are trying to knock-down a gene using siRNA /
mock siRNA in a stably transfected cell line(parental), which is also ur
control cell line (which u are treating with only lipofectamine)
knockdown is more in control cell line!! than proper siRNA
actually knockdown is present in all transfected cell lines!!
your control cell line no longer a control cell line.
any contamination in lifofectamine? or optiMEM?
try chaning the tranfection agent..
On Thu, Apr 24, 2008 at 2:43 PM, Kamalian, Laleh <L.Kamalian from liverpool.ac.uk>
> Hi everyone
> I have come across this problem. I have stably transfected my cells using
> Lipofectamine 2000 as the transfection reagent. So far, I have been using
> my parental cells as the calibrator during transient transfection. Now for
> the stable transfection I have used scrambled si RNA oligonucleotide to
> transfect my cells and used a pool of these cells as my control. Using
> quantitative PCR to measure the mRNA of the colony cells and the control
> cells, my colony cells show about 70% gene knock down comparing to the
> scrambled si RNA control cells, which is good. However comparing to the
> parental cells, the control shows about 90% less expression of the gene of
> interest. Once before during my transient transfection I had the same
> problem when used "no DNA" as my negative control i.e. Lipofectamine without
> any DNA. At that time I had used western blot to measure the protein
> instead of QPCR. I had noticed that my negative control lysate had
> considerable decrease in protein expression. My question now is can
> Lipofectamine or transfection condition cause the gene knock down? It
> especially doesn't make sense now that my scrambled RNA control cells have
> been growing in the selective medial for at least 4 weeks now. I am
> thinking that even if the transfection condition had affected the cells
> transiently, it can't effect the mRNA level after being eliminated from the
> growth media. Am I right or am I missing something in here?
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