Generating hpRNA? - how does one create a reverse complement? (Jun/09/2005 )
I've been fascinated by RNA silencing for a while now and I've finally had the opportunity to integrate it into my research. I would like to create hpRNA from a full-length cDNA (532bp). I'm working with plants so I don't have to worry about the interferon response that is associated with > 30bp dsRNA's. How would I go about generating the reverse complement of that full-length cDNA? I want to use the full-length cDNA so I don't have to worry about which siRNA will be most efficient.
Basically, this is what I need to create:
5'-...CGT ACC GTA TGC...---InterveningSequence---...GCA TAC GGT ACG...-3'
------(full-length cDNA)------------------------------------(reverse complement)
After transcription, I should have:
5'-...CGU ACC GUA UGC...---
3'-...GCA UGG CAU ACG...---
Is it possible/cost-effective to have a 532bp DNA fragment synthesized?
Although it's possible to synthesize a 0.5 kb sequence, it would be more costly than constructing 2-3 short hairpin siRNA vectors. If you randomly pick a siRNA target site on your gene you will have over 80% chance for silencing the gene. If you use two shRNA constructs the chance will be even higher. So which is better?
Ahhh, so there is no special trick then. I'll definitely look into that. Thanks for the help!
Since you are working with plants, then longer ds RNA will be more appropriate, and then you should do in-vitro transcription of RNAs. It is too expensive to synthesize long dsRNAs
T7 is best:
1) have your cDNA clone with T7 in both directions
2) have two cDNA clones: one to IVT sense strand, one to IVT antisense strand.
Thanks for the reply. I'm already in the process of performing option 1 that you described and I plan on using my full-length cDNA (532bp). Hopefully all works out. I'll post with details later since I'm trying a few shortcuts that I think will work out great.