miRNA inhibitors - 2'-O-methyl versus Ambion antisense oligos (Oct/05/2006 )
I am just beginning to look at miRNAs and how they might regulate a couple of proteins that I am interested in. Does anyone have experience with miRNA inhibitors, such as 2'-O-methyl oligoribonucleotides, or Ambion's miRNA inhibitors, or other products? I am looking for the following information: cost, efficiency in knocking down the mature miRNA expression (a percentage of wildtype), stability of the inhibitor (meaning either half-life or for how long a period a knockdown of 70% of the miRNA is seen after initial transfection or electroporation). Does one inhibitor work better than the other in certain cells (i.e. adherent v. suspension, primary v. immortalized)? Any help or experience would be greatly appreciated. Thanks.
-Zach
-Zach
Hi!
I am just back from the RNAi meeting in Prague where I got some nice information about miRNA inhibitors.
Some companies put some mystery around miRNA inhibitors (eg. Ambion). It is not true. Just have a look at the current litterature (Tuschl's group article in Nature for example) and you will see that these inhibitors are simply antisense oligos. As any other antisense oligo, miRNA inhibitors should be stable and non toxic for the cell line you are using.
In this context, 2' O-Me modified oligos are perfect for that purpose.
Some may introduce additional modifications to further enhance the stability (such as phosphorothiaote linkages). This works fine too. Others are using LNA bases. The problem with LNA concerns the design. So far, nobody has been able to predict correctly the effect of the addition of LNA into an oligo. In addition when you buy an LNA antisense from Exiqon they never give you the sequence information. So you never know what you are using!
Finally, I saw an interesting poster in Prague describing the positive effect of increasing the length of the antisense oligos against miRNA. These guys showed that targeting only the mature works fine but extending the length of it (so adding some bases from the pri-miRNA) improves the results.
For more info, contact me directly at d.poncelet@eurogentec.com
Regards,
Dominique
So, how did you guys assay the activity of your anti-mir/antisense oligo?
Specially, if the target of your miRNA is unknown.
Specially, if the target of your miRNA is unknown.
I've a collaborator who uses a reporter plasmid with a near-complementary miRNA target in the 3'-UTR sequence of the reporter gene. Introduction of the miRNA into cells bearing this expression plasmid causes translation repression and a decrease in the reporter signal.
While you are considering antisense structral types for miRNA knockdowns, look into my company's product: Morpholino antisense oligos. They are not degraded in cells (though they are diluted by growth) and they have no sequence-independant toxicity, in contrast with oligos bearing toxic phosphorothioate modifications.
Let me know how I can help.
- Jon
Here's a recent publication showing Morpholino knockdowns of miRNA:
Flynt AS, Li N, Thatcher EJ, Solnica-Krezel L, Patton JG. Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. Nature Genetics 2007; 39: 259 - 263.
- Jon