The gene you want to knockdown may be so essential for the survival of the astrocytoma cells.  So you may be knocking down the gene below a give threshold resulting in cello death.  You may have to switch to a regulated system like a TET-inducible system.

Dear Amira,

Both Frederick and Daniel are correct. Indeed toxicity can be a side effect of gene silencing when :

- transfection reagent is toxic

- concentrations of siRNA and/or transfection reagent are not optimized

- innate immunity of the cell is triggered: cells sees the siRNA transfection as a viral or bacterial attack and go under apoptosis. This phenomenon is observed with some transfection reagents that trigger immune pathways and lead to the produciton of interleukine and chemokine.

- incubation time with complexes (a medium wash may be necessary)

- siRNA targets also some gene essential for cell survival (in this case siRNA design is the cause)

- others (cell health, siRNA purity....)

The first option would be to use a fluorescently labelled siRNA to confirm that conditions are ok,

The best option to my point of view would be to try other reagents such as Lullaby (http://www.ozbiosciences.com/transfection-sirna/24-lullaby-sirna-transfection-reagent.html) or another method of transfection such as Magnetofection with SilenceMag (http://www.ozbiosciences.com/transfection-sirna/65-silencemag-magnetofection-reagent-cell-silencing.html) or NeuroMag, dedicated to neural cell types and primary neurons (http://www.ozbiosciences.com/transfection-cell-specific/52-neuromag-neuron-transfection-magnetofection.html).

would you like to try our transfection reagents, please do not hesitate to contactme directly at: [email protected]

good luck with your experiments,

best regards,

Cedric