RNA structure requirement for drosha-DGCR8 - (Nov/01/2007 )
anybody knows what does microprocessor drosha-DGCR8 need to process pri-miRNA?
I want to overexpress miRNA , the less genoimc sequences cloned the better. So I wonder what the least sequence flanking mature miRNA is nessecery, in the condition that drosha-DGCR8 can cut the primary transprict well.
Am I clear? Thank you!
I followed the recommendations of Ambion for cloning into their vector (even though I didn't use their vector) and cloned between 50 - 100nt of flanking sequence on each side of the miRNA. I obtained good expression of the miRNAs that I cloned in this manner. I don't think anybody knows the exact sequence requirements.
DO you mean eather side should leave 50~100nt? By the way,50~100nt from mature sequence or pre-miRNA?
I donn't want the unecessary sequence to be cloned .ha
Yes, each side should have between 50 and 100nt flanking sequence. When I did this, it was from the pre-miRNA. Why are you so concerned about the extra sequence? It's not too much extra, and if you cut it too short there is the possibility that it won't be expressed at all. Alternatively, you could consider taking a look at the miR-30 based shRNA constructs. Just make a shRNA with the miRNA sequence and clone it into the vector that has the miR-30 flanking sequence. Check out PMID: 16141338 and PMID: 16200064.
OK, Thank you very much! Then I should clone appromixly 200~300bp into vector
I think the extra sequence may complicate the expreesion system
Could please answer the other question of mine about how to check the complete transcript of a particular miRNA?
I'm not sure what other question you mean. I did qRT-PCR (TaqMan) to detect transcription of my miRNA, you could also try a Northern. Both would show transcription of the miRNA.