siRNA electroporation - (Aug/31/2009 )
Hi everybody,
We recently bought an electroporator (Gene pulser, Biorad) and we plan using it on regular basis mainly for siRNA transfections. We are using C2C12 cell line. We want to optimize the transfection efficiency by monitoring GAPDH-cy3 labeled siRNA luptake by flow cytometry. We first tried unsuccessfuly with FAM-labeled siRNA until we understood that the electroporation quenches the FAM dye. We bought the siRNA-cy3 from Ambion and they recommend using 5nmol. However, this would be the amount of siRNA recommended when doing straightforward or reverse transfection, using transfection reagent present in the media with the cells for 24h. Someone told me that if we do siRNA electroporation, due to the fact that pores are formed in the cell membrane for very short time, we need to increase the siRNA amount significantly. If someone has experience with this, can you recommend what concentration of siRNA to begin with. Also, should we do more than one pulse? What voltage you normally use? I realize that this is very cell specific, but any information you can give me, would be useful. Thank you in advance!
Hi,
I use amaxa nucleofection for siRNA delivery and my experience is that more siRNA is needed. I use 100 uM stock and add 1-3 ul/1 million cells.
I had to test various buffers and settings for the cell line I use before I got round the one that works.
Hope this helped, good luck,
KRisztina
biolog on Aug 31 2009, 02:40 PM said:
We recently bought an electroporator (Gene pulser, Biorad) and we plan using it on regular basis mainly for siRNA transfections. We are using C2C12 cell line. We want to optimize the transfection efficiency by monitoring GAPDH-cy3 labeled siRNA luptake by flow cytometry. We first tried unsuccessfuly with FAM-labeled siRNA until we understood that the electroporation quenches the FAM dye. We bought the siRNA-cy3 from Ambion and they recommend using 5nmol. However, this would be the amount of siRNA recommended when doing straightforward or reverse transfection, using transfection reagent present in the media with the cells for 24h. Someone told me that if we do siRNA electroporation, due to the fact that pores are formed in the cell membrane for very short time, we need to increase the siRNA amount significantly. If someone has experience with this, can you recommend what concentration of siRNA to begin with. Also, should we do more than one pulse? What voltage you normally use? I realize that this is very cell specific, but any information you can give me, would be useful. Thank you in advance!