Protocol Online logo
Top : Forum Archives: : siRNA, microRNA and RNAi

Generating hpRNA? - how does one create a reverse complement? (Jun/09/2005 )

Hello,

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...---
-----------------------------------InterveningSequence
3'-...GCA UGG CAU ACG...---


Is it possible/cost-effective to have a 532bp DNA fragment synthesized?

Thanks,
Hank

-haringsh-

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?

-pcrman-

Ahhh, so there is no special trick then. I'll definitely look into that. Thanks for the help!

-Hank

-haringsh-

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

or

2) have two cDNA clones: one to IVT sense strand, one to IVT antisense strand.

-Cali Girl-

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.

-Hank

-haringsh-