I am currently doing cell experiments with GFP linked miR plasmids to overexpress microRNAs. I have done reciprocal experiments using anti-miR microRNA inhibitors from life technologies but I am looking for an alternative to knockdown a microRNA using a plasmid that can be grown in bacteria preferably with a fluorescent reporter. The main goal is to allow us to do more of these knockdown experiments without the expensive cost of the anti-miR products.
I found one of the most useful methods to inhibit miRNA is using activatable Sensor Oligonucleotides. its efficient and specific with clear and clean background than using a plasmid.
Hello David,
When you say GFP reporter, do you intend it to report the level of miRNA expression in the cell as well? If not then my answer might be useful to you.
While Duaa has made an excellent point with the sensor Oligos which are quite effective, I decided to move on to sponges a couple of years back.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957044/
Take a look at this review which covers some of the Key points of Sponge usage. They also denote some of the major disadvantages and the most interesting thing they mention is the inducible system.
Basically what you have to do attach a GFP linked sponge against your desired miRNA to a TeT inducible promoter in a lentiviral construct like pLVTHM and make a cell line out of it in the cell of your choice. This cleans up the endogenous population the miRNA in question quite effectively when you induce the expression with TeT and the GFP gives you proof of expression of your sponge.
It's one of the sure fire way of inhibiting the activity of your miRNA and the best way to check for it is to use either a luciferase reporter or WB against the primary target protein of the miRNA.
Hope this helps.
Best,
Roy
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