For getting successful co-transfection, you need high quality DNA, a validated siRNA against the gene of interest and log phase growing HEK293 cells. The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection:
1. To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, transfection can be performed at same time by using one reagent, such as Lipofectamine 3000 or Lipofectamine 2000.
2. To study the effect of siRNA knockdown of an endogenous gene, siRNA transfection should be done first using an RNAi specific delivery reagent such as Lipofectamine RNAiMax. 4-48 hours after siRNA has been delivered, DNA can be transfected using Lipofectamine 3000 or Lipofectamine 2000. The time of post-transfection delivery of DNA may need to be optimized.
Once the appropriate method of delivery is determined based on the application, transfection should be optimized for DNA, siRNA and lipid delivery reagent doses. For transfecting HEK293 in 96-well, we typically use these amounts per well:
• DNA: 0.1-0.2 ug
• siRNA: 1-3 pmoles
• Lipid reagent: 0.1-0.3 ul