RNA Interference - how to produce siRNA (Jan/27/2004 )
Hi there,
It seems to me there are many strategies for making siRNA for RNA interference and also there are different ways of introducing siRNA into cells. What strategy is the most successful one?
Thank you for any hints.
GG
I don't know of the easiest but I just read of a system that is much cheaper than the usual. Its MessageMuter shRNAi from Epicentre. I plan on giving it a try soon.
I am going to try the pSUPER retrovirus vector , which can stably express shRNA transcript in target cell line. have anybody tried this?
Hiya,
I heard that retroviral vectors are no good for long-term stable expression - does anyone have any comments to this?
Cheers,
Cath
I use the Ambion pSIlence 3.0 vector but It doesn't work well,I don't know the reason
It seems that Ambion produces many fancy things but few of them really work.
I know someone using Ambion and they've had a lot of success - but it seems very variable - 3.0 will work for some shRNA in some cells types and 2.0 will work better in others...they bought both and try both vectors for every shRNA. (U6 is supposed to be the better promoter for RNAi)
cat
Depends what you want to study.
Any DNA-based method of siRNA expression - viral or plasmid-based - will kick off certain cellular stress responses when the siRNA is expressed, which seems to be a consequence of the processing required to turn an shRNA into an active siRNA. If you're studying stress-response pathways this is obviously bad news, plus engagement of stress-response pathways will have knock-on effects on the expression of other genes. If you don't care about this then you could give plasmid-based siRNA expression a try (transient or stable, your choice).
I don't like any strategy that involves chopping up RNA into 'siRNA' (Ambion will sell you kits that do this) because you have no real idea what it is that you're transfecting into your cells. Even with chemically synthesized siRNA we've found massive differences in the cellular response depending on who we buy them from and - importantly - the level of purity they're supplied at. Specifically, Qiagen offer a service that promises '~90% purity' of the siRNA - we tried it 'cos it was cheap but went back to the more expensive options pretty quick. Just wasn't worth the waste of time/effort/reagents/money. Bottom line: The cleaner the better.
I use chemically synthesized siRNA for two reasons. 1) Publications to date suggest strongly that they don't tend to kick off the stress-response pathways that shRNA do, and 2) you know exactly what you're introducing to your cells. Plus, for transfection/silencing controls defined chemically synthsized siRNA is probably the best to control for an active RNAi process (an siRNA that silences an non-essential gene product) as well as a non-active siRNA (scrambled control, or a sequence that has no target in your cells). It's a trade-off 'cos if you get lucky and design an effective siRNA you can use it for years before your stocks run out, but it's an expensive way to screen for effective siRNAs. Plus, this method means that you have to put your cells through ~4 hours of serum starvation as part of the transfection process, whether this is acceptable or really bad news for your experiments is up to you to decide.
I would like to share this article from The Scientist which offers some tips on how to start RNAi study.
http://www.the-scientist.com/article/display/18847/
I have tried the pLITMUS28i vector supplied by NEB. Easy to clone a sequence and transcibe dsRNA in vitro using the T7 promoters on both sides of the MCS. Then you can transfect the product to your celkl line using Lipofectamine 2000 or a reagent of your choiice. You might need to digest with Dicer. I used it to inject RNAi in crustaceans to check for antiviral response. Was quite easy.