siRNA, shRNA are attached to the mature mRNA so if you want to attach a RNAi with an intron, it won't work.

siRNA, shRNA are attached to the mature mRNA so if you want to attach a RNAi with an intron, it won't work.

Mmmh, strongly doubt you can succeed. RNAi works through the RISC, which is assembled in the cytoplasm. Normally RNA arrives already spliced to the cytoplasm... although there are some exceptions and cytoplasmic splicing as a regulatory mechanism of gene expression is known. But in very particular cases only!! You should at least first try a cytoplasmic RNA extraction and a RT-PCR using primers on the intron you are interested and on a subsequent or preceding exon to verify the presence of the unspliced RNA in the cytoplasm... But you should also determine what is the percentage of this RNA form compared to the spliced, possibly with a RT-qPCR... easily, even if you are lucky enough to find the unspliced form in the cytoplasm, it will only be a minor fraction... so your RNAi will most likely not be effective...

Is there a reason why you wouldn't use the 3'UTR sequence? In plants it usually diferentiates well between members of the same family.

Dear all, these are excellent tips. I am already planning on doing a RACE to sequence the transcripts from this gene, and possibly get the full length primary transcript.

I have only determined the putative transcripts based on aligning RNASeq data from various sources to the genome, and determined the putative level of expression based on microarray data. Thus confirming the transcripts and level of expression more specifically is the next step.

I particularly like this approach of using the 3'UTR, Nada, will take a closer look into it as soon as I have it well determined, thanks!

3'UTR are clearly a better bet, it would work on any model.

No!

5'UTR is often GC-rich, thus is not a good region for a successful RNAi. 3'UTR may offer good target sequences for a successful RNAi. However, some regions of 3'UTR bind to RNA-binding proteins, which may reduce the efficiency of RNAi.

Have you considered using morpholinos to avoid the splicing of your target intron?

In this way you could be introducing a stop codon signal or you could target this intron with a siRNA once it is retained.

Slipping in from my experience with miRNA (micro-RNA), I'd agree that the 3'-UTR is a good target to go for. Natural miRNA molecules target the 3'-UTR of a gene to downregulate its expression. That being said, I don't know if arthropods have miRNA. It might be worth investigating, if you have infinite funding, infinite time, and infinite grad students--that's always the problem.

Not siRNA, but you can use antisense oligos with some stable modifications (Eg. 2-Omethylated) to target the very beginning of an intron. This should cause exclusion of the upstream exon in the mature RNA.

Note: you want skipping of the upstream exon to cause a frame-shift and create premature stop codons

May be this paper could help: Tyson-Capper AJ1, Europe-Finner GN. Mol Pharmacol. 2006 Mar;69(3):796-804.

Morpholinos, or other antisense approaches could work as above, but I suspect much less attractive in the context of ants. There is clearly a dsRNA uptake mechanism in ants, and quite likely an amplification aspect (common to many organims) that makes RNAi the approach most likely to succeed on a whole-organism basis. If you were working on cells then antisense approaches might be best. 3'UTR is the obvious target, I agree.

Why not design short LNA inhibitors unique to each of your sequences?

Check this out, it may give you ideas: http://www.sciencedirect.com/science/article/pii/S0965174812001464

Nope, but you may be able to hit the 3'UTR and many have already suggested. Here's another thing to think about: Do any of the splice junctions generate unique regions post-processing? I did similar work targeting human IgLCs had to adopt that strategy for similar reasons.

No its not work for intron region . Better designed the siRNA taking end of 5'UTR and start of exon.

As already mentioned, it will not work because the siRNA targer the mature mRNA. What you can do is a siRNA that target a unique exon junction of your splicing variant.

Not sure how to deleiver your dsRNA? transgenic ants expressing a hairpin RNA/miRNA or feeding dsRNA or injection?

as I know it won't work ... and 3'UTR and 5'UTR regions sometimes are not as well specific as we want .... hmm ... that's the idea: design a siRNA for two exon just like designing the exon-junction primers or as Rakesh Ranjan Said use 3'UTR/exon or exon/5'UTR to increase the specificity...

siRNA targeted to introns would only work in those infrequent cases of alternative splicing involving intron retention

The answer is simply no.

Eduardo,

As other people have said, iIt is very unlikely that targeting the intron will work since splicing is usually too quick for siRNAs to have a chance to act and RNAi mostly functions in the cytoplasm, at least in mammals; not sure about ants. You could try creative tricks to get specificity. For example if the related genes you want to avoid silencing have a different exon-intron structure than your gene of interest, then make "hyrbrid" siRNAs across the introns of your gene of interest. In other words, make siRNAs with complementarity with two exon termini.

Dear all, these tips were all fantastic, many many thanks. The technology of LNA is entirely new to me, and the recent review on insect miRNA is certainly a must-read. This hybrid approach to exon combinations is really a very good approach and also combining exons and UTRs. This is all very useful advice, many many thanks!

Dead Eduardo Goncalves Paterson Fox,

Were you able to carry out the knockdown by targeting introns? Do you have any recommended paper were this was done?

Also as suggested by Luis Franco, I think this can inhibit proper splicing thereby affecting matured mRNA formation. I am looking at a group of siRNA I believe that they are targeting introns in a vital gene having a weird alternative splice pattern and not really sure what to make of it yet. please let me know,

Thanks

The LNA GapmeRs available now through QIAGEN are very successful when starting with a primary mRNA sequence. You can even specify the intronic regions for the GapmeR targeting.