6 replies to this topic

[Samantha]

Hi,

What system do you use to knock down miRNA expression in mammalian cells? I know some use custom design 2-O-methyl RNA oligos (antisense of the mature miRNA), miRIDIAN hairpin inhibitors (Dharmacon), anti-miR microRNA inhibitors(Ambion), LNA oligos, etc. How's the knockdown efficiency of each method? Is there any one better than the others?

For the custom design 2-O-mehtyl RNA oligos, all the bases are 2-O-methyl modified?
Thanks a lot!

Samantha

[pcrman]

We have tried virtually antimiRs from all companies. We found LNA antimirs are quite impressive in knocking down miRs.

I believe every base is 2'OM modified in oligo antimirs.

[Samantha]

pcrman, on Jun 16 2009, 09:22 PM, said:

We have tried virtually antimiRs from all companies. We found LNA antimirs are quite impressive in knocking down miRs.

I believe every base is 2'OM modified in oligo antimirs.

Thanks pcrman. Do you design the LNA antimirs by yourself or directly order from commercial companies such as Exiqon? What transfection reagents do you use?? Thanks.

Samantha

[Jon Moulton]

Samantha, on Jun 16 2009, 07:13 PM, said:

I know some use custom design 2-O-methyl RNA oligos (antisense of the mature miRNA), miRIDIAN hairpin inhibitors (Dharmacon), anti-miR microRNA inhibitors(Ambion), LNA oligos, etc.

Morpholinos are a good option.  Their per-base affinity is lower than the LNAs, so they have better sequence specificity, good enough for use in embryos (which are exquisitely sensitive to teratogenic off-target RNA interactions).  They don't use the phosphorothioate linkage, so they avoid the toxicity and oligo-protein interactions associated with that linkage.  In addition to directly targeting the guide strand of an miRNA, Morpholinos can be used to inhibit miRNA maturation.

Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development.  PLoS Biol. 2007 Jul 24;5(8):e203 [Epub ahead of print]

There are more citations at the bottom of this page:
http://www.gene-tools.com/node/31

Best wishes for your knockdowns!

[Neanderthal]

Dear Members,

Thanks for sharing your knowledge.

I was wondering whether there is any freely available tool that will help us to design antimiRs/antagomiRs. Please hasre if you know of any such tool/software/site.

Thanks
K.

[kaveh]

Neanderthal, on 14 March 2012 - 01:38 AM, said:

Dear Members,

Thanks for sharing your knowledge.

I was wondering whether there is any freely available tool that will help us to design antimiRs/antagomiRs. Please hasre if you know of any such tool/software/site.

Thanks
K.

The design is simple. You just want to have a sequence complimentary to the sequence of your microRNA. As if they want to form a double helix. If your microRNA sequence is GCGCAAACU then your inhibitor is going to be CGCGTTTGA. Now, it's your choice to pick a DNA, modified RNA or LNA to synthesize that sequence.

[Jon Moulton]

You can design the oligo slightly longer than the guide strand, extending into the loop or past the Drosha cleavage site into the stem.  This improves the oligo affinity for the pre-miRNA/pri-miRNA and helps in inhibiting maturation of the microRNA.  This is a standard strategy for Morpholino design targeting miRNAs and was originally explored in:
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development.  PLoS Biol. 2007 Jul 24;5(8):e203 [Epub ahead of print]
http://biology.plosj...al.pbio.0050203

This strategy is also useful if there is sequence within the guide strand that would require a stable self-complementarity in the oligo sequence.  The oligo target can be shifted farther into the flanking sequence to break the self-complementarity.  An oligo targeted in this fashion will not bind well to pre-processed miRNA on RISC but can be a very effective inhibitor of miRNA maturation.