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What's the most easiest way to perform RNAi - I'd like to apply the technique (Jul/04/2005 )

I have never used RNAi, but I do know that it is very powerful and useful in research. I plan to use this techniqe in my own research, but I konw only a little in this realm. Could anyone tell me what's the most easiest and convient way to perform RNAi in research in cell biology? Your reply will be greatly appreciated.

-babygogo-

Hi,

The easy and quick way of doing RNAi is using synthetic siRNAs to transfect cultured cells. You need at least two siRNA duplexes for each gene and a control siRNA which lacks homology to any sequence of your organism.

There are quite a few companies providing siRNA synthesis and transfection reagents such as Invitrogen, Ambion, Dharmacon, Qiagen.

You may not even need to design siRNAs, because those companies offer pre-designed or validated siRNAs for most human genes and genes of other species.

Transfection is not a big deal, but you may need to find a particular transfection reagent that works for your cells.

Good luck.

-pcrman-

Dear pcrman, thank you very much for your reply. I believe that using synthetic siRNAs is the most easiest one. However, it is too expensive for me and I really cannot afford that. My boss will not give me a chance to try. I heard that there are some vectors for siRNA, and all we have to do is to make a construct and then transfect. I don't konw whether it will work all the times in this way.


QUOTE (pcrman @ Jul 4 2005, 10:48 AM)
Hi,

The easy and quick way of doing RNAi is using synthetic siRNAs to transfect cultured cells. You need at least two siRNA duplexes for each gene and a control siRNA which lacks homology to any sequence of your organism.

There are quite a few companies providing siRNA synthesis and transfection reagents such as Invitrogen, Ambion, Dharmacon, Qiagen.

You may not even need to design siRNAs, because those companies offer pre-designed or validated siRNAs for most human genes and genes of other species.

Transfection is not a big deal, but you may need to find a particular transfection reagent that works for your cells.

Good luck.

-babygogo-

Unless someone is going to give you a hsRNA vector, I don't think constructing your own hsRNA will save you much. Let's make a comparision.

Suppose you are going to knock down one gene and not for stable transfection.

If you use synthetic siRNA you will need 2-3 siRNAs (two gene specific and one control) which cost around $600 (20nM scale). Each siRNA can be used for at least 200 transfections (for 6-well plate at the concentration of 50 nM, a very high concentration).
Total cost: $600
waiting time: 1 week (different suppliers vary)
Interferon response: less likely


If you are going to build your own hsRNA, you will need

at least 6 54-mer oligos ($15x6=$90)
Vector system: $300-$500
competent cell $$
transformation reagents $$
plasmid extraction $$$
clone identification $$
maxiprep $$
sequencing $$
Time: 1 week, 1 month, 3 months... (depending on how lucky you are)
Interferon response: very likely

-pcrman-