I am new to this site. I am in a big problem. I am starting to do siRNA using Lipofectamine 2000 and I am lost. I am working with L6 cells. Could anyone help with a protocol? Thank you very much.
You can get the protocol from the company`s website. But you need to find out the optimal ratio of the lipofactomine and siRNA, cell density. Different cells lines have different optimal conditions.
Here are some protocols and an article that my help you out...
Invitrogen. Transfecting siRNA into Mammalian Cells Using Lipofectamine 2000.
Lipofectamine 2000 has been used successfully to transfect short interfering RNAs (siRNA) into mammalian cells for RNA interference (RNAi) studies (Gitlin et al., 2002; Yu et al., 2002). This reference provides general guidelines and procedures to transfect siRNA into mammalian cells using Lipofectamine 2000. Suggested transfection conditions are provided for a number of mammalian cell lines as a starting point. If you are using these cell lines or another mammalian cell line, we recommend optimizing transfection conditions to obtain the best results for your target gene and siRNA.
Transfecting and Plating RAW 264.7 Cells with Lipofectamine 2000. AfCS Procedure Protocol PP00000182
Version 1, 10/31/03
The following procedure is for simultaneous transfection and plating of RAW 264.7 cells. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when using pEYFP-N1 from Clontech.
Brian Dalbyl, Sharon Cates, Adam Harris, Elise C. Ohki, Mary L. Tilkins, Paul J. Price and Valentina C. Ciccarone. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods
Volume 33, Issue 2, June 2004, Pages 95-103
Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposomeDNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs.
Thank you very much. I will check those references.
Please feel free to post whether or not any of these methods were helpful. It would be very beneficial for the rest of the board to hear your reaction and/or results.