Boba155 - 23-5-2011 at 19:42
Hey, not sure if this is the place to ask, but I'll hope that someone has experience in this and give it a shot.
I want to try in-vivi shRNA post growth knockout of genes. However, I believe this will require quite a bit of shRNA to transfect an entire animal.
Does anyone know how I could go about producing large amount of a specific shRNA sequence? I can't really afford to pay 30K for a simple project such
as this 
.
Thanks!
simba - 24-5-2011 at 11:11
I think this should be in the biochemistry board...In fact, I think this doesn't even belong to this forum, this is a chemistry forum, not genome
research forum.
ssdd - 24-5-2011 at 11:31
Having worked in a lab which deals with shRNA, siRNA, and dsRNA I can say that this is not an easy task. You will find it near impossible (without the
proper equipment) to rid your supplies of RNase A which will make for major problems. Also, you must recall that RNA is not the most stable of
molecules and will be easily degraded (that extra OH group makes it much more reactive than DNA). Lastly, you mentioned transfecting an animal with
this RNA sequence, depending what you are doing you may run into legal and ethical issues beware of that.
As to a synthesis of an RNA strand this is going to be exceedingly difficult to anyone without the proper equipment. The only 'easy'
way about this would be to amplify the RNA from an isolate, but this is also difficult: http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_3.html
Given all of this I would not recommend a project such as this to the home scientist, it will only result in frustration and cost more than it could
possibly be worth.
ssdd - Tim
Saerynide - 25-5-2011 at 08:37
Custom synth from Sigma is pretty "cheap". I had a sequence available commercially that I needed, and they were able to make it for me at less than
half the price the vendor was selling it.
Of course, this "cheap" refers to the having-a-shitton-of-funding kind of "cheap"