How to increase RNAi efficiency by siRNA transfection? - (Jul/12/2006 )
I am trying to achieve knock down in a cell line derived from an SV40 transgenic mouse. I have tried a couple of different delivery systems (HiPerFECT and Lipofectamine2000) without getting more than 25% transfection effeciency (as measured by FACS of a FITC conjugated scrambled sequence). I have mainly concentrated in altering the siRNA concentration without much effect on effeciency.
What would people suggest I do next, what is the order of preference of optimising the transfection (ie. concentration, reagent concentration, time course, cell density etc)?
Any help or advice would be greatly appreciated.
When you say you estimated your transfection efficiency, did you mean that you transfected cells with a labeled siRNA and liposome then checked for the number of cells that carry the label by fluorescent microscopy? In this case, you should keep siRNA constant and vary the transfection agent, because if you do the other way around your signal intensity would be different.
I had the same problems with a cell line. I used Oligofectamine (Invitrogen) and I solved the problem!
It might also be helpful to keep the siRNA conc. the same while changing the amount of transfection reagent you're using. Dharmacon recommends 100nM siRNA or less for efficient silencing.
Thanks for the tips, I will try altering the liposome concentration and keeping the siRNA concentration constant. I have previously tried up to 100uM siRNA concentration and haven't seen any toxicity, I have been told by someone in another lab that concentration will kill the cells as anyone experienced this. If this doesn't work than I might take dnafactory advice and change reagent again.
Also when I was measuring transfection effeciency I was doing using the Fluescent Activated Cell Sorter (FACS) and measuring the number of cells above background not looking at overall fluorescence so altering the siRNA concentration should not effect the analysis. Although it is generally true that increasing the concentration lead to increased mean fluorescence the percentage of cells transfected remained constant.