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synthetic microRNA stability


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#1 mahrak

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Posted 02 September 2013 - 11:12 PM

hello everybody

 

Here is my situation:

Instead of using synthetic double stranded mature microRNA from ambion, I am going to have both guide and passenger strands chemically synthesized. then I am going to use annealing buffer and annealing conditions from Metabion to get double stranded mature microrna. then i am going to use a transfection reagent( Effecten) to introduce microrna to cells.

 

1- has anyone been in the same situation before?

2- are micrornas prepared this way stable enough?

3- what chemical modifications can i use to increase the stability? would adding FAM/2'OM to 3' end change the stability or RISC involvement?

 

 

All the best

 

 

   

 



#2 pcrman

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Posted 02 November 2013 - 08:56 AM

1- has anyone been in the same situation before?

Yes, that is doable. 

 

2- are micrornas prepared this way stable enough?

Once  double-stranded, they are pretty stable. 

 

3- what chemical modifications can i use to increase the stability? would adding FAM/2'OM to 3' end change the stability or RISC involvement?

What do companies do to synthesize miRNAs is to chemically modify the star strand to suppress its potential activity to reduce offtarget effects. The commonly used chemistry is 2'F and 2OMe. The modification on the star strand applies to several nucleotides, not just one end. If you want to also modify the guide strand, you have to run some test to make sure the modification does not impair activity. 






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