hello
I just have the sequence of precursor-miRNA and the organism I am working on doesn't have its genome published. I looked for several days and couldn't find it.
So my question is whether using the precursor-miRNA and cloning it into pcDNA vector will overexpress it after transfection into fish cell lines.
Or should I add flanking region?
Thanks
Hi Adham,
Yes! you need approximately 60 bp surrounding the pre-miRNAs (flanking region) which includes single strand-double strand (SD) junction.
Cheers
Ali
thanks for the answer. the major problem for me it is the organism doesn't have a genome database and the sequencing of its genome not yet out . the organism is olive flounder so in miRBASE.org i found just the pre-miRNAs sequences. so it is not possible to know the flanking region of the pre-miRNA.
You could design primers to extend from the known pre-miRNA region and sequence the surrounding region to determine the flanking sequence.
thanks for your answer.i have just the 70-80 bp of pre-miRNA so not easy to do RACE. can you give a paper or a protocol about this procedure?
I am not sure if this could help, did you check if it is conserved between hsa-miRs or mmu-miRs .. If it is you could try getting the upstream and downstream sequences flanking your pol-pre-mir from UCSC genome browser.
The mature microrna are conserved some of them but when I blast the precursor sequence it doesn't give a good results.
If transient transfection is the main purpose, you can just design or order mature miRNA mimic. I guess you already know the mature miRNA portion in your pre-miR. If you want to clone mature miR, you should choose some shRNA expressing vector under the control of PolIII promoter (pSuper etc). Order oligos of sense and anti-sense strand, anneal and clone it into vector. Good luck.
thanks Amin for your answer. my main object is to use the pre-microRNA and overexpress in fish cell line. so if i use pSuper vector it will be expressed as shRNA not as microRNA.
Your purpose is not clear. Do you want to study the processing of pre-microRNA into mature microRNA? or just overexpression and see its effect on target gene? If just overexpression, I had that experience of designing it as shRNA vector and measured the overexpression of mature miRNA by stem-loop RT pcr. It works as mature miRNA. You can also found similar procedure in the following paper:
http://www.ncbi.nlm.nih.gov/pubmed/23032974
Both shRNAs and pre-miRNAs undergo Dicer processing and RISC loading in a similar fashion anyway. I don`t know whether fish biology is more complicated than human biology though.
thank for your help. can you please provide me with the protocol for designing shRNA as microrna and it is possible to use pSilencer as expression vector?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA