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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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co-transfection
Posted by: airtight (IP Hidden, Unregistered user, )
Date: April 27, 2005 12:39AM
Hi All,
I am doing RNAi experiments and so I did the following cotransfections The first time, I cotransfected my siRNA, luciferase reporter and beta-gal vector. The siRNA is targeted at the luciferase gene and beta-gal is used to control transfection efficiency. After transfection, I found my siRNA had great effect on how much reproter and beta-gal vector enter the cells. I got very low luc activity for cells transfected with siRNA, which may simply indicate the fact that siRNA compete for lipo with other vectors instead of RNAi effect. Although some publications adjust the amount of lipo used for the amount of total DNA or RNA in the transfection mixture, but most don't. I seriously doubt the published results that the observed lower reporter activity is caused by RNAi. In order to overcome this problem, I did a two-step transfection. First, I transfect luc-reporter vector and then split the cells for another transfection with siRNA and beta-gal which is used to normalized transfection efficiency of siRNA. In this way, siRNA won't affect reporter transfection. However, again, siRNA and beta-gal compete for lipo and the more siRNA is used, the lower beta-gal activity I got. Thus, I cann't normalize transfection efficiency using beta-gal activity because siRNA and beta-gal compete with each other instead of entering the cells proportionally. Based on these observations, now I would say that it is totally unpredictable what is going on if you throw in more than one vector into the cells. Do you agree?
Re: co-transfection
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: April 28, 2005 06:15PM
Do siRNA with Lipo on one tube (A), and beta-gal with Lipo on another tube (B), let them form DNA/lipo complex separately. Then, do co-transfection with different ratio of (A) and (B) on your luciferase-transfected cells to see the effect.
Re: co-transfection
Posted by: hutman (IP Hidden, New member, 5)
Date: February 18, 2006 09:26AM
Much easier and cheaper way is to transfect cells by electroporation, the competition is excluded. We do it routinely as described using nucleofection with GTporator solution. This way we have typically over 90% transfection efficiency with all common cell lines (HeLa, HEK293, CHO...). Look here
[www.gentrend.cz] Hutman
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