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.
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.
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.
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.
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