Which shRNA provider or calculator do YOU use? - (Jan/27/2009 )

Hi!

I'm really confused with the different possibilities designing siRNA, in my case shRNA.
Last time we purchased sequences at ambion, with the result that only one out of 5 produced a significant knock-down. To confirm this knockdown I need a second functional siRNA,
but I'm having difficulties deciding which option to take:

ordering again at ambion, or maybe dharmacon?
Which provider is the best one for shRNA?
Or should I use a freely available calculator?
And if so - which one?

It would be so very nice, if you could share your experience with me

elisa

Hmm, I thought that ABI/Ambion has a list of validated siRNAs? Dharmacon also has siRNAs, I think thay have the deal where you order 4 and at least 1 is supposed to knockdown by at least 70%. My understanding of shRNA is that you should validate the siRNA to knockdown your gene first, then you can prepare the shRNA construct.

miRNA man on Jan 29 2009, 11:29 AM said:

I have used and extensively tested MANY siRNAs from many companies and find Dharmacon to be the best. The others are also acceptable, but I find the Dharmacon ones to more consistently give higher levels of knockdown.

For shRNAs, I am only just starting to use them but I would NOT reccomend Open Biosystems pSM2 based ones as their knockdowns are reported to be quite awful and only a few actually work at all.

Mountainman on Jan 30 2009, 12:31 PM said:

Hi,
I also don't recommend pSM2 from open biosystem as they are not efficient at all

well, as for me, Open Biosystems saved my ass... I tried to knockdown Prion gene expression and using LMP retrovector with shRNAmiR cloned from pSM2 vector I got 90% downregulation in MEL and NIH3T3 cells and approximately 80% in CAD5 cells.