Transfection efficiency with RNAi - help (Jul/14/2006 )

Hi-

I'm trying my first siRNA experiment and well despite my strong urging, my boss has refused to buy ANY control reagents: NO negative control, NO control to target a different protein, and NO fluorescent duplex to assess transfection efficiency.

I've been told that siRNA's are a bit easier to transfect than plasmids into cells. I have plasmid transfection conditions already worked out for my cell line. I know that every cell line is different but do you generally find that the conditions that are optimized for plasmid transfection are optimal for RNAi as well? Or will I have to reoptimize? I hope not because I have no way of assessing transfection efficiency independent of knockdown without a fluorescent duplex.

If anyone has any advice, it would be greatly appreciated, as without this fluorescentl duplex, every optimization experiment will take me about a weeks time (transfection 24-72hrs + Western for protein). If it helps, I will be using lipofectamine 2000 on a relatively difficult to transfect schwann cell line.

Thanks very much,

Mountainman

I can only suggest to try transfecting siRNA as u would with ur plasmids ( I am sure u would have planned it anyway). Have a non transfected group as control to compare knockdown.

Its hard to when u dont have any positive control for transfections.

Good Luck !!!

Agreed with scolix. Co-transfection with a reporter plasmid (EGFP) will give you a low end of estimation of transfection. You will use much less amount of siRNA, such that the precise optimal ratio maybe very different for siRNA than DNA. However, if you do it with your plasmid, since siRNA only accounts for minute part of the total nucleic acid that you are delivery, you will be ok.

I guess your PI just wants to see what you can get, before getting too fancy. Once you have some good indication, you can go ask for more. Cheer up.

OK thanks for the help you two,

I guess I'll start with the conditions for plasmid transfection and work from there. Sorry, I guess this post was pretty much equally helping me vent as getting technical help. I guess I was just a little frustrated with my boss' decision ehehe. It makes sense I suppose, as we are a bit short on funds.


Thanks,
Mountainman