combined overexpression and knockdown experiment - (May/03/2007 )
Dear all
I am planning to transiently overexpress a certain proten (PROTEIN A) in a cancer cell line, at the same time I want to knockdown another protein (PROTEIN 
in the same cell line that transiently overexpress(PROTEIN A), and this genetic manipulation will be followed by drug treatment for 24hrs. the point is that the cell line becomes confluent within 4 days, so I should do overexpression and knockdown within the same day. for overexpression, lipofectamine is used but for siRNA oligofectamine is used. My proposed protocol will be as follows: perform knockdown first and after 6 hrs of siRNA transfection remove the medium (optimem) and replace it with fresh optimem then start overexpression procedures and incubate overnight. on the next day replace optimem with complete medium and add drug treatment for additional 24 hrs.
Do you think that this protocol will work?
any suggestions are welcome
thanks
I am planning to transiently overexpress a certain proten (PROTEIN A) in a cancer cell line, at the same time I want to knockdown another protein (PROTEIN
in the same cell line that transiently overexpress(PROTEIN A), and this genetic manipulation will be followed by drug treatment for 24hrs. the point is that the cell line becomes confluent within 4 days, so I should do overexpression and knockdown within the same day. for overexpression, lipofectamine is used but for siRNA oligofectamine is used. My proposed protocol will be as follows: perform knockdown first and after 6 hrs of siRNA transfection remove the medium (optimem) and replace it with fresh optimem then start overexpression procedures and incubate overnight. on the next day replace optimem with complete medium and add drug treatment for additional 24 hrs. Do you think that this protocol will work?
any suggestions are welcome
thanks
Important point, Can you get sufficient knockdown in the given time frame? try a simple knockdown to quantify its effect and the time frame. Depending on that make changes or try it out as proposed.
I am planning to transiently overexpress a certain proten (PROTEIN A) in a cancer cell line, at the same time I want to knockdown another protein (PROTEIN
in the same cell line that transiently overexpress(PROTEIN A), and this genetic manipulation will be followed by drug treatment for 24hrs. the point is that the cell line becomes confluent within 4 days, so I should do overexpression and knockdown within the same day. for overexpression, lipofectamine is used but for siRNA oligofectamine is used. My proposed protocol will be as follows: perform knockdown first and after 6 hrs of siRNA transfection remove the medium (optimem) and replace it with fresh optimem then start overexpression procedures and incubate overnight. on the next day replace optimem with complete medium and add drug treatment for additional 24 hrs. Do you think that this protocol will work?
any suggestions are welcome
thanks
Important point, Can you get sufficient knockdown in the given time frame? try a simple knockdown to quantify its effect and the time frame. Depending on that make changes or try it out as proposed.
Yes, the sufficient KD should be checked, but also I am afraid that leave cells in Optimem for so long may not bee very good for cells, esp if you want to do drug treatment right after. Too much stress. I think that the lipofectamine can be used also for siRNA although not as efficient. But otherwise, you may want to try using other transfection agent that allow the use of normal medium for protein A transfection, e.g. Effectene (QIAGEN), Transfectene (BIORAD). Both are good reagent and require much less DNA compared to lipofectamine, moreover, it can be added into normal medium so there is no need to change medium. We have found that the Enhancer in the kit of Effectene is quite efficient (and you always finish it WAY later compared to Effectene itself, so you always got spared of Enhancer) and can be used with other reagent like Lipofectamine to increase the efficiency of transfection.