This protocol has been standardized in HUVECs but can be applied to other cell types or suspension cells.  Data on siRNA delivery using Targefect F-2 plus Virofect has been kindly provided by Dr Michael Potente, Department of Cardioology, University of Frankfurt, Germany

1. HUVECs used were from  from Cell Systems
2. the culture media is EBM with 10% FCS and supplements (Singlequots)
3. HUVEC should be 80-85% confluent at the time of transfection
4. Be default we use 60 mm dishes for cell culture, therefore, the following protocol is designed for 60 mm wells

Conditions:

-          add 500 ul DMEM (Optimem works as well) first

-          add 18 ul of the desired siRNA (20 uM) and mix

-          add 12 ul of F2 and mix

-          immediately add 25 ul of Virofect and mix

-          incubate for 20 min at 37°C

-          in the meantime replace the culture media with 2 ml of fresh EBM 10% & suppl.

-          add siRNA complexes dropwise to the cells

-          change culture media the next day and assay for gene expression silencing after 24, 48 and 72 hours

Very recent data suggest that you can actually reduce the siRNA amount by half without losing any silencing effect. As you can see from the attached western blot, the silencing efficiency seems to greatly depends on the target sequence of the siRNA and not so much on the transfection method per se as all three methods gave comparable results. In our hands HUVECs work very well for the siRNA approach and we routinely get silencing efficiencies about 80 to 90% using different target genes. Nevertheless, the Virofect protocol is very easy to handle, suitable and time saving.