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HUVEC siRNA transfection help please - I need suggestions on what else to try! (Jul/02/2008 )

Hi,
I am trying to transfect siRNA into HUVEC cells. So far, nothing.

Briefly, I feed my HUVEC's with fresh complete medium for 1 hour before transfection.
They are approx 70% confluent (in a 6-well plate).
I use 100pmol of siRNA in 250uL Optimem with 5uL of Lipofectamine 2000 in 250uL of Optimem. These are mixed as per the Lipofecatmine 2000 protocol.
I replace the complete media with 1mL of optimem and add the complexes.
Incubate for 5hrs and change back to complete medium.

I have tried, different concentrations of siRNA and Lipofectamine, different cell densites and transfecting in complete medium. Nothing.

If anyone has any other suggestions or can shed any light on where I am going wrong it would be much appreciated.
The student in the lab before me was able to do it easily, so it is making both me and my boss VERY frustrated that I can't get it to work!

Thanks in advance.

-than4-

QUOTE (than4 @ Jul 2 2008, 07:15 AM)
Hi,
I am trying to transfect siRNA into HUVEC cells. So far, nothing.

Briefly, I feed my HUVEC's with fresh complete medium for 1 hour before transfection.
They are approx 70% confluent (in a 6-well plate).
I use 100pmol of siRNA in 250uL Optimem with 5uL of Lipofectamine 2000 in 250uL of Optimem. These are mixed as per the Lipofecatmine 2000 protocol.
I replace the complete media with 1mL of optimem and add the complexes.
Incubate for 5hrs and change back to complete medium.

I have tried, different concentrations of siRNA and Lipofectamine, different cell densites and transfecting in complete medium. Nothing.

If anyone has any other suggestions or can shed any light on where I am going wrong it would be much appreciated.
The student in the lab before me was able to do it easily, so it is making both me and my boss VERY frustrated that I can't get it to work!

Thanks in advance.

See if you find some good protocols here:
http://search.vadlo.com/b/q?sn=158621799&a...ction&rel=0
..

-cellcounter-

Why dont you try starving the HUVEC cells in serum free medium ( serum free medium +0.1% BSA) for few hours and then do your siRNA transfection.
I hope this helps. For me it works all the time.
Tulip

-tulip-

For siRNA transfection I use siRNA transfection reagent from Santa Cruz either with transfection medium or with EGM2 medium (Lonza) WITHOUT serum. after 7 hours just change the medium and add fresh complete medium.
Another way is to use FUGENE, you just have to find you siRNA working concentration, then just follow the specs.
Hope it helps, both ways work with me.

-Fizban-