SiRNA in MIN6 cells, nucleofection - (Sep/11/2007 )
Im am very new with the SIRNA technic and so is my lab.
As the lab next door did few i have been asked to get closer to them and ask question. I started my experiment regarding to their advices, but now facing certain problems especially regarding the transfection method.
We use nucleofector kit from amaxa. I have been told i should use a "carrier" plasmid (wich is not really a carrier as its Double strand DNA) so that the electric charge of my solution would allow my ds to get into the cell.
So far i read lots of article and none of them mention so.
Cause the way they do their protocol they use 5µg DNA total (200nM or my DS rna completed up to 5µg with my plamid in that case pUC19)
My concern is that i have lots of cell death i know that electrporation is really stressful for the cells , but now I am starting to wonder if that amount of plasmid that i co-transfect (plasmid who has strictly no reporter use or something equivalent) is not toxic for my cells.
Any help comment would be appreciated.
in our lab we only use Amaxa when we want to transfect a lot of cells. We never use a carrier plasmid. For 24-well or 6-well plates we use DharmaFECT1 reagent, which works really well for us. It gives very low cell death and very high knockdown (typically between 80-95%).
We have tried other transfection reagents as well (Lipofectamin, Oligofectamin, FuGene,...) but none of them gave sufficient silencing in our MIN6 cells.