RNAi Transfection Protocols

Life Technologies has the most complete collection of transfection reagents with exceptional performance for the delivery of DNA, siRNA, Stealth™ RNAi and BLOCK-iT™ RNAi Vectors, in traditional or difficult-to-transfect cell lines. Remember to refer to the Seven Steps to RNAi Success when planning RNAi Experiments.

General transfection protocols

• Transfecting Stealth™ RNA or siRNA into Mammalian Cells using Lipofectamine® RNAiMAX
• Cotransfecting Plasmid DNA and synthetic RNAi into Mammalian Cells Using Lipofectamine® 2000
• Reverse Transfection of Stealth™ RNAi or siRNA using Lipofectamine® 2000 in a 96 well plate format

Cell-type Specific Transfection Protocols for Stealth™ RNAi and siRNA

“Customers also found Lipofectamine® RNAiMAX transfection reagent to work well in the additional cell types. Click here for details.

Stealth™ RNAi and siRNA transfection concentrations
The transfection concentration of a Stealth™ RNAi or siRNA duplex is determined by dividing the number of moles of siRNA used by the final volume of the transfection (i.e. starting medium volume + transfection mixture volume).Using a 24-well plate we typically transfect 0.5-5 pmol of siRNA in a 100ul transfection mix to 500ul of medium in each well to give a 1 to 10nM final concentration.The certificate of analysis for Stealth™ RNAi or siRNA has instructions on making a 20µM stock solution. If transfecting in triplicate make 400µl of 5x transfection mix by adding 0.1-1 ul of the 20µM stock to 399µl of Opti-MEM®. Then add 100µl of this transfection mix to each of your wells.20µM Stealth™ RNAi or siRNA = 20pmol/µl

Cell line database
Invitrogen provides many free cell line-specific transfection protocols in the Cell Line Database.The Database is intended to enable researchers to easily find references related to the use of Invitrogen products and cell lines of interest. This database is by no means exhaustive, but a collection of data dependent on the efforts of Invitrogen Technical Service.

Co-transfecting a vector and siRNA:
When co-transfecting plasmids and siRNA, use the transfection conditions appropriate for a plasmid transfection. The siRNA component usually adds negligible nucleic acid mass and can be ignored. Find more about the transfection conditions and references for your cell line in the Cell Line Database.

Troubleshooting transfection experiments
Invitrogen has created a general table to help troubleshoot transfections using cationic lipids. In addition we have compiled a list of frequently asked questions about transfections.