siRNA in vitro transcription-Ambion kit? - (Jan/26/2006 )
I plan on doing some siRNA. I have 2 transcripts I would like to knock out so if I was to use chemical synthesis the cost would be in the region of about £1000 (based on the siRNA each costing approx £120, you are advised to try 3 for each target and would also need a negative control for the one showing best effciency (8X120).
This is very expensive so I have been looking at the in vitro transcription kit from Ambion which is £440 and can be used for 15 reactions.
Has anyone any experience of this kit? Anything good or bad to say about it? Or has anyone used any other kits which they would recommend?
If you buy the in vitro transcription kit at £440, it is not guaranteed to succeed. There are many technical hurdles during the process and the resulting siRNA may not as potent as synthetic siRNA.
If you can find in the literature that someone has designed working siRNAs against your gene, why don't you use it. Recently I wanted to knock down 3 genes and don't have enough budget for 3x3 siRNAs. So I found a paper from Tuschl group which has worked on the 3 genes. Actually they listed 3 siRNAs for each gene in their paper, I just picked one for each gene. Luckly, the single siRNA works beautifully for all three genes.
Thanks for your reply. Looking in the literature is a good idea but unfortunately its not a described gene I am trying to silence. I totally agree synthetic siRNA would be a better option but the costs seem very high but, at the same time, I have yet to hear anything positive and convincing about the in vitro transcription method !
i have used that kit and works fine (90% silenced), either transfecting the RNA duplex with liposome-based reagents (lipofectamine 2000) or electroporating, depending on the cell line.