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Troubles to transfect siRNA
Posted by: Fei Wu (IP Hidden, New member, 2)
Date: March 28, 2005 10:04PM
I am transfecting siRNA of Sp1 into MCF-7 cells on the first day using Lipofectamine 2000, on the second day, i need to transfect my reporter gene-luciferase and beta-gal into MCF-7 cells using GeneJuice. Then on day three, i will harvest the cells to measure my reporter activity. But I have the problem that every time, when I transfect with siRNA, it will cause my beta-gal activity decreases a lot.
What's the reason could cause my siRNA transfection effect my beta-gal expression? thanks.
Re: Troubles to transfect siRNA
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: March 29, 2005 12:55PM
One possibility is that Sp-1 site also on the promoter of beta-gal reporter. Does luciferase reporter have the same promoter as beta-gal??
Another will be indirect effect of knockdown Sp-1 on transcription of beta-gal reporter.
Re: Troubles to transfect siRNA
Posted by: Fei Wu (IP Hidden, New member, 2)
Date: March 29, 2005 06:01PM
Yes, that's right, that's maybe the problem, since beta-gal also has GC-rich sequence on its promoter, where Sp1 may bind.But i don't think it can cause so much effect. Even though Sp1 may bind beta-gal promoter. But there is no available information to show that Sp1 can activate beta-gal expression. And on the other hand, my colleague who is doing the same experiment, doesn't find such decrease of beta-gal activity. I am thinking what kind of mistake i may make to cause beta-gal activity decrease so much.
Thanks for your reply. Fei
Re: Troubles to transfect siRNA
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: March 30, 2005 11:55AM
You both used the same siRNA (same sequence, same amount, same transfection on same cells??)?? If yes, something must be wrong in different hands.
Sp-1 is a strong activator as well as coactivator-recruitor (recruiting other co-activators), without it the signal you saw is only basal level of transcription.
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