I am trying to co-transfect DNA and siRNA into HEK cells. Can you give me some advice on how the most effective way to do this.
This question is part of the following Ask The Expert session:
Transfection Optimization for Improved Efficiency and Performance
Company: Life Technologies
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