HELP: transfection reagent for suspension cell - mouse hybridoma siRNA transfection (Dec/14/2005 )
Hi, does anyone here has positive experience of transfecting siRNA or shRNA into suspesion cell?
The cell I'm heading for is mouse hybridoma, which is originated from B lymphocyte.
I've tried four different reagents, Lipofectamin 2000 (Invitrogen), FuGene (Roche), HyperFect (Qiagen) and siIMPORTER (upstate). With fluorescein-labelled negative control siRNA, I could see vague fluorescent signal from some of the transfected cell. The positive control siRNA targeting GAPDH is from Ambion, however, qPCR found no difference of GAPDH expression level between transfected cell and control cells.
No serum, no antibiotics was supplied during the transfection, FBS was supplied 6hrs after the transfection.
Sorry for the wordy post, thanks for any suggestions and comments.
I am currently having the same problem. I have tried transfecting human B cell lymphoma cell lines using a variety of transfection agents with little success including:
DMRIE-C (InVitrogen), siPORT amine (Ambion), siPORT NeoFX (Ambion), siPORT Lipid (Ambion), HiPerFect (Qiagen), Trans-IT TKO (Mirus).
By flow cytometry, there is a shift using fluorescently labelled siRNA but under the microscope these look like dying, granular cells. I too have been using Ambion's GAPDH siRNA as a positive control.
For suspension cells, Amaxa Biosystems Nucleofection system seems to work or retroviral transduction. For my purposes, however, I need to avoid viral and electroporation methods. I'm interested in other people's comments.
Thanks for the comments. Feels better that someone else suffering the similar pain,
Had some talk with Qiagen and Ambion tech support, got the suggestion of using amaxa stuff and electroporation, respectively. I have the same feeling about electroporation. And even if I'm willing to try amaxa system, my cell is not in their cell line database.
Anyway, guess we have to keep trying. and need some luck,