looking for a good siRNA transfection reagent for 3T3 L1 cells ? - (Mar/06/2006 )

Hi ,
I tried RNAiFect (Qiagen) to transfect siRNA (synthetic duplex) to 3T3 adipocytes. it doesn't wrok well. Can anyone suggests a better procedure. Our lab used same method for Hela cells and it works well. I saw some publications indicated siRNA delivary to 3T3 cells by electroporation? Differentiated adipocytes seems to be difficult to transfect!
I am new to teh forum. Thanks for your help.
Arcella

Hi Arcella,
i think you can try with lipophilic reagents as lipofectamin 2000 (invitrogen) (it's a very easy system) or try to change method, so that you can use virus (i.e., lentiviral transfection)

if you'll try let me know if you have a good goal!

Thanks for the advice. I am working on lipofectamine 2000. When I transfected with different expression vectors, such as pEGFP-N1, I had trouble getting them differentiated. Hope lipofectamine will work.
Arcella

Hi everybody,
Recently we have purchased Amaxa nucleofactor machine for transfection both plasmids and siRNA.
In our hand it worked well. It is excellent even in two hours some cell surface markers appear after transfection of freshly isolated lymphocytes It is expensive. It does not allow common reagents to use. The company has their own proprietry solutions and protocols already built in in to the machine. For any new cell line you have to optimize your protocol. You will get help from the company. they are very helpful. on the flip side, the machine is expensive. If you have any questions, contact me.

Actually, before my arrival to this lab they have purchace this neoucelofactor instrument, and I see it sitting aside. It seems they haven't had good cell survival using it for differentiated adipocytes, besides when I read the protocols for this system, it looks like needs lot of siRNA which is expensive. I am worried to trypsinized and collect differentiated adipocytes which may reduce survival in addition to electroporation shock. If I fails this lipofectamine 2000 procedure I will try it. At least I am happy to hear it works for another cell type. Thanks.
Arcella

Hi,

You can try Deliver-X transfection reagent from http://www.panomics.com/deliverx_sirna.cfm for siRNA transfection.
It works fine with me.