Antibiotics during siRNA transfection
Posted 25 January 2005 - 09:27 PM
Can anyone tell me if the presence of antibiotics in the media before and after transfection affect its efficiency? I've been using oligofectamine for siRNA transfection in adherent cells. I use complete growth medium (i.e., with antibiotics) to grow the cells prior to transfection and also in the 3X-serum-medium after the 4h incubation with the siRNA-oligofectamine complex. Though I get a not-too-bad level of transfection I'm wondering if my use of antibiotics is hampering efficiency. Suggestions anyone?
Thanks a bunch!
Posted 25 January 2005 - 09:42 PM
Posted 31 January 2005 - 08:30 AM
i don't use antibiotics in the mixture siRNA (or ADN) / oligofectamine.
I don't know if it is necessary for you to use antibiotics, but as i know, antibiotics are recommended to be added to days after transfection.
In a transfection, i let the mixture adn + oligofectamine on the cells overnight and change it for a fresh non selective media the day after the trasfection.
If you get too much toxcicity, you cant add a 2Xserum non selective media Three hours after starting the transfection and put a complete fresh media the day after the transfection day. I guess this reduce toxcicity bu not transfection efficiency
All results have been made by analysing the transfection of a linearized plasmid containing the ORF of a fluorescent protein and observation of ration fluorescent cells/total cells on a fluorescent microscope.
For siRNA experiments done without antibodoes and target protein level analysed 24h and 48h post transfection.
Posted 31 January 2005 - 03:56 PM
If it's the former, then it might just be the siRNA you're using, some don't give massive knockdown but this can be fine if it gives you a strong phenotype. You might just need to try another siRNA sequence. Also, you won't get 100% knockdown with any siRNA as there will be pools of sequestered mRNA within the cell that the RNAi machinery can't reach. And I wouldn't use antibiotics at any stage, you don't need them - I plate cells for transfection in antibiotic-free media the day before transfection, and feed 'em 4 hours after transfection (I use Oligofectamine, too) with medium+3xserum but no antibiotics...works just fine. You have to remember to add L-glutamine to your medium+3xserum to a final concentration of 12 mM, though, so that it ends up at 4 mM final concentration when you add it to your transfections (reason: L-glutamine is so labile I always assume that in any bottle of media I buy, it's already gone off by the time I recieve the media...if you're using something like OPTI-MEM which you don't get through very fast, I 'm pretty sure this may be the case, as I have had very dodgy transfection results when I've forgotten to add L-glut!).
If it's that your cells just aren't transfecting that great then you're just going to have to fiddle about with transfection reagents (some work better with certain cell types than others), or your cell platings (clumped cells transfect poorly, you ideally want a monodispersed resuspension of cells before you plate 'em), or your cells are too confluent when you're trying to transfect them (again, you just gotta determine this empirically for each cell line by trying transfection at different confluencies), or try assessing transfection efficency (you could try a GFP reporter but I've found something like FITC-Dextran - from Sigma - is much better for this). Plus, some cell types just won't transfect and/or silence very well.
Basically, it just depends on what you're trying to achieve with your transfections. We find that even the most co-operative (cancer) cell lines, which are usually very happy to take up siRNA, only do about 80% transfection efficiency under optimal conditions, with mRNA/protein knockdown (by qRT-PCR and Western blotting) averaging ~60-70%...but this gives very strong phenotypes and/or huge knock-on effects on other cellular protein.