So confused with RNAi - (May/05/2009 )

Hey all,

Just wondering if any of you loverly people can help me. I am going to be starting RNAi experiments soon to remove the expression of a gene that i want to put back into my cells. Will this work? how would i go about designing the siRNAs and also could i target the promoter for this gene and prevent transcription?

Thanks in advance guys, I know its a long one,

Bex

Kami23 on May 5 2009, 08:37 AM said:

RNAi is used to reduce the level of mRNA for your desired gene. Rather than designing one yourself, I would first try some of the verified siRNAs available from several companies (Dharmacon, Ambion, QIAgen, ABI, etc.). One of them is sure to have an siRNA for your gene of interest. If you must, there are a variety of websites that will design them for you if you input your target sequence. Again, siRNAs are generally used to target mRNA, resulting in their degradation by the dicer complex. If you want to reintroduce your gene of interest, you must make sure that your transfected construct is sufficiently mutated (silent mutations, of course) that its transcripts will not be recognized by your siRNAs; however, please note that this is not as easy as it sounds as siRNAs can target mRNA with several mismatches with no problem. Sometimes the best option is to use a different species of your gene of interest, such as a human siRNA and reintroduction of a rat gene expressing plasmid.

As for targeting the promoter itself, several recent Science papers have shown that siRNAs can also be designed to target promoters for genes that are actively being expressed (since the chromatin in these areas is "breathing" allowing entry of your siRNA), but this is not a typical role for siRNAs by researchers nor will it work for genes that are not constitutively expressed and the controlling promoter regions must also be known.

Thanks Dr Teeth,

That was the answer i was hoping for because it proves me right We will have to use a different approach.

thanks again

Bex

If you are interested in doing an RNA rescue experiment, this is fairly straightforward with a steric-blocking oligo. You target the oligo to the 5'-UTR of the gene you want to knock down. This prevents translation by halting the initiation complex as it travels from the 5'-cap to the start codon, so the mature ribosome does not form and therefore the RNA is not translated. By cloning the coding region of the gene of interest into an expression vector that has a different 5'-UTR (so the construct lacks the oligo target), you can express RNA which will not be affected by the presence of the oligo; if coinjection of this rescue RNA with the steric-blocking oligo returns the cells/organism to a wild-type state, you have shown specificity of knockdown (which is the typical goal of this experiment).