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Optimizing transfection of miRNA


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#1 sgylee

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Posted 06 February 2010 - 09:43 AM

Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much

#2 Fizban

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Posted 09 February 2010 - 09:21 AM

Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much


can't help without details....what is the problem here?
why are you stuck?
what do you mean when u say 80nM mirna? single strand synthetic? overexpressing vector? commercial mimics?
try to help us helping u! :)
Fiz

#3 j123

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Posted 10 February 2010 - 08:53 AM

Try NeoFX transfection reagents from AB. It works very well for me. It also depends on different cells. Good luck!


Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much






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