miRNA seems to be at the center of translational regulation for many proteins. To study this phenomena there are many commercially available miRNA inhibitors as well as miRNA mimics. After transfection with one of these it should be possible to measure changes in the translation of some target protein - in theory. I have found that when using miRNA inhibitors, which are small sequences of RNA themselves and bind to the miRNA to block them from interacting with message RNAs, that the transcript levels of my miRNA actually goes up! Is this some kind of transcriptional feedback due to the loss of function of the miRNA? Does this mean that if I use miRNA inhibitors I will only be chasing my tail as the cell just spits out more of the miRNA to compensate? Mimics are an alternative, but how do you measure their effectiveness if your target protein is usually not translated in the first place?
Hi Michael,
If you want to block a specific miRNA, hairpin inhibitors are a great route (try Dharmacon or Exiqon LNA inhibs). The problem you may be having is that the inhibs may actually be stabilizing the miRNA transcript or that there may be a homeostatic response as you mentioned.
What you need to do though is to verify if miRNA FUNCTION is knocked down. If there aren't any good validated target genes to verify by qPCR I would definitely look into getting luciferase reporters that contain the miRNA seed sequence. miRNA inhibition should lead to increased reporter levels. Genecopoeia and SwitchGear Genomics both sell miRNA luciferase reporters.
Good reading material - http://www.thermoscientificbio.com/uploadedFiles/Resources/mimics-inhibitors-tech-note.pdf
**If you want to block ALL miRNA biogenesis there is always the option of transiently knocking down Drosha or Dicer levels by siRNA.
Michael
Your data looks extremely interesting... This could be a compensatory effect of the cell that reacts to miRNA inhbition with a feed-back mechanism.
Regarding the method, I personally do not like small inhibitors. I prefer miRNA sponges, because they are more stable and you can obtain better yields of inhibition.
Good luck... Cheers
Paco
Alexander - Sorry, I don't have any info on screening programs. The miRNAs I'm after were reported to target my genes/proteins of interest in another author's paper. I'm trying to validate the finding in my own cells as it may have implications for my work. I'm kind of new to miRNA.
Francis - Thanks for the heads up on sponges, I'll give those a try perhaps after the next round of trials.
Hi Michael,
If you want to block a specific miRNA, hairpin inhibitors are a great route (try Dharmacon or Exiqon LNA inhibs). The problem you may be having is that the inhibs may actually be stabilizing the miRNA transcript or that there may be a homeostatic response as you mentioned.
What you need to do though is to verify if miRNA FUNCTION is knocked down. If there aren't any good validated target genes to verify by qPCR I would definitely look into getting luciferase reporters that contain the miRNA seed sequence. miRNA inhibition should lead to increased reporter levels. Genecopoeia and SwitchGear Genomics both sell miRNA luciferase reporters.
Good reading material - http://www.thermoscientificbio.com/uploadedFiles/Resources/mimics-inhibitors-tech-note.pdf
**If you want to block ALL miRNA biogenesis there is always the option of transiently knocking down Drosha or Dicer levels by siRNA.
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