Hi Eugene,

One option would be to try another transfection reagent. I can suggest

http://www.ozbiosciences.com/transfection-cell-specific/28-insect-cells-dna-transfection-flyfectin-reagent.html

It has been successfully used on S2 cells as shown by many publications such as this one: http://www.ncbi.nlm.nih.gov/pubmed/24469638

If you want to try it let me know at [email protected]

I can send you a sample

Good Luck

Olivier

Maybe this is not relevant or technically challenging, but a different possibility would be to use dsRNA tagged with a fluorophore. This is, for instance, routinely done with siRNA transfected in human primary cells. Thus, by having a transfection reporter, you could in principle isolate positive cells to have a "pure" population independently of the transfection efficiency.

Hi Stefano, unfortunately I do need this protocol later for a wide screen so it is quite nopractical solution( 

Hi Eugene, 

Have you successfully used S2 RNA bathing with a different construct?  Also, I use Life Gibco Schneider's Medium (21720-024) with HI FBS (10%) and penstrep. I get 75-90% knockdown for most genes.  How big is your dsRNA construct?

Hi jeffrey, my Rna construct ±500bp, so should be in range. Could you describe what protocol do you use? 

I use the same amounts of RNA as the Rogers paper in a slightly larger volume (10ug/1.5mL).  For some genes I target 2 regions and add 10ug of each construct.

Hi, Jefferey. But Rogers does use serum free medium (sf900) not Schneider's Medium with FBS... Do you also repeat treatment each 24h?