siRNA failure - Knock down failure with oligofectamine (Jun/15/2006 )

Hello
I try to transfect macrophagic cells by siRNA and I don't arrive to see a dimunition of the expression. The manner with which is done the coupling step of the oligofectamine with the double strand of RNA (pipetage or soft agitaion) is so crucial?
Thank-you for your assistance

-rymaxs-

QUOTE (rymaxs @ Jun 15 2006, 07:47 AM)

Those are difficult-to-transfect cells. Maybe electroporation is better way to go.

-genehunter-1-

do you have an optimised transfection procedure for these cells? Gene hunter has the right point.
does the sequence you use have been published/validated?

-fred_33-

QUOTE (fred_33 @ Jun 16 2006, 02:41 AM)

do you have an optimised transfection procedure for these cells? Gene hunter has the right point.
does the sequence you use have been published/validated?

Hi
infact thes sequences that I have choosen for proceeding RNA interference are validated and
tested with the same macrophagic cells , but I have diffulties to achieve gene silecing with oligofectamine reagent
in addition after transfection I observe cell mortality
thank you for your help

-rymaxs-

QUOTE (genehunter-1 @ Jun 15 2006, 01:22 PM)

QUOTE (rymaxs @ Jun 15 2006, 07:47 AM)

Those are difficult-to-transfect cells. Maybe electroporation is better way to go.

Hi
I have beforehand transfect these cells by electroporation and I succed but I have done it with plasmids not with siRNA. I think that it's not possible to proceed gene silencing by electroporation method
thak you for your idea

-rymaxs-

hi rymaxs,

can you post your oligofectamins lot number please? all of your story sound s somehow familiar.

thanks

tobi

-Tobikenobi-