Optimizing transfection of miRNA - Need help on transfection using lipofectamine 2000. (Feb/06/2010 )
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
-sgylee-
sgylee on Feb 6 2010, 06:43 PM said:
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
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
-Fizban-
Try NeoFX transfection reagents from AB. It works very well for me. It also depends on different cells. Good luck!
sgylee on Feb 6 2010, 12:43 PM said:
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
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
-j123-