[Arabidopsis] Fwd: Re: Questions on plant RNAi

Dr. James J. Campanella via arab-gen%40net.bio.net (by james.campanella from montclair.edu)
Thu Aug 30 06:49:58 EST 2007


>Date: Wed, 29 Aug 2007 10:36:36 -0500
>From: MICHAEL L SULLIVAN <mlsulliv from wisc.edu>
>Subject: Re: Questions on plant RNAi
>To: "Dr. James J. Campanella" <campanellj from mail.montclair.edu>
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>Original-recipient: rfc822;james.campanella from montclair.edu
>
>Jim,
>
>I wish I could offer you information on this. I haven't really tried 
>too much silencing of specific genes of a multigene family, and my 
>silencing strategies (using the pHannibal hairpin vector) have been 
>mostly driven by cloning considerations. I do believe I came across 
>a size range in one paper I read that said 200-700 bp fragments were 
>appropriate, although I've also used bigger (1100) successfully. 
>Most "information" I've heard is very anecdotal. From some of these 
>accounts, I gather it is actually very hard to predict what will 
>work to silence either an entire family or an individual member. On 
>the other hand, somebody once told me that you could silence a 
>specific gene that differed from another by only one or two base 
>pairs by designing only a very small ~22 bp RNA that included the 
>difference. This guy might have been basing it more on theory though 
>than on actually having done it.
>
>My impression is that in the end this ends up being largely 
>empirical. To silence individual members of a gene family, I think I 
>would first try to target the 3'UTRs, assuming they are not highly 
>homolgous, or parts of the genes that share the least identity and 
>lack extensive stretches of homology (i.e., I'd avoid including any 
>part of the gene that had more than 10 or 12 contiguous bases of homology.
>
>Sorry I can't offer more information. Hopefully some researcher out 
>there have better answers.
>
>Mike
>
>
>----- Original Message -----
>From: "Dr. James J. Campanella" <james.campanella from montclair.edu>
>Date: Wednesday, August 29, 2007 9:18 am
>Subject: Questions on plant RNAi
>To: arab-gen from magpie.bio.indiana.edu, 
>plantbio from magpie.bio.indiana.edu, methods from oat.bio.indiana.edu
>
>
> > Dear Colleagues,
> >
> > I have several questions about practical plant RNAi to which I have
> > been unable to find the answers in the literature. I suspect that
> > these questions are mostly naive, but I since none of my colleagues
> > here on my own campus work with plant RNAi, I thought I may be able
> > to find the answers out here.
> >
> > First, are there any good practical guides or reviews out there on
> > plant RNAi? Most of the literature that I have been able to dig up is
> >
> > on animal RNAi which is quite different in terms of RNAi probe design
> >
> > and activity. So far, the most practical advice has come from the
> > Methods in Enzymology volume on RNAi.
> >
> > Second, again, there is a great deal of literature on designing the
> > short RNAi probes for animals. There are even computer programs to
> > help design those short sequences. However, I have been unable to
> > come across any advice on choosing the long (300-500 bp) sequences
> > needed for plant RNAi.
> >
> > Third, I am working with a family of plant genes, and I want to be
> > able to knock down specific members of that family. What is the
> > breakpoint percentage of homology at which you need no longer worry
> > about cross-over inhibition against homologues? If the probe is 50%
> > homologous to another family member which is not the target, will you
> >
> > get knockdown of the homologue? 40%? 30%? Is this even common
> > knowledge, or does it come down to finding out these answers by
> > practical experiments with your own species and gene family?
> >
> > Thanks for the help,
> >
> > Jim Campanella
> >
> >
> > James J. Campanella,
> > Associate Professor,
> > Department of Biology and Molecular Biology
> > Montclair State University
> > 1 Normal Avenue
> > Montclair, NJ 07043
> >
> > Alternate email address: jcamp from alumni.uchicago.edu
> >
> > Ph: 973-655-4097
> > Fax: 973-655-7047
> >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods

James J. Campanella,
Associate Professor,
Department of Biology and Molecular Biology
Montclair State University
1 Normal Avenue
Montclair, NJ 07043

Alternate email address: jcamp from alumni.uchicago.edu

Ph: 973-655-4097
Fax: 973-655-7047 



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