Stable miRNA transfection - (May/12/2008 )
Is there any kit or procedure out there for stable miRNA transfection for over-expressing miRNAs endogenously in vitro? Similarly, is there any way of stably knocking down miRs?
Stable transfection is possible like for any other gene once you have a proper construct inside the proper vector.
overexpression in this way is quite easy to achieve, but i have no experience in stable knock down
For stable knockdown you could construct a miRNA sponge (Ebert et al Nat Methods 2007).
Thank you both for your responses... I have a vector I can use (pSilencer from Ambion) but any idea how you would actually make the DNA fragment with the pri-miR in it? Does a company construct this for you?? Sorry for being an idiot, but this is totally new for me so in front of my boss I pretty much look like this when he's talking about it.
You typically PCR-amplify the pre-miRNA. To get this I find the miRNA hairpin sequence and amplify with an extra 100bp either side. Stick enzyme sites in the primers at the 5' end and you're good to go (hopefully )! I think that System Bio have ready made plasmids with miRNAs inside if you want to go in that direction...
You're awesome miRNA...very helpful...I just have a few more questions. Do you need to do a blast search to find the extra 100bp flanking each side of the hairpin? AND...what source of DNA do you use to amplify from...does it matter? Also, what is System Bio, is that ABI?
Here is the link: http://www.systembio.com/miRNA_Precursor_Collection.htm
To get the sequence I just use the ucsc database...search for your microRNA of interest , using the "miRNA" track in the tables section. Output-sequence, everything else as default. then you want genomic DNA and in the next window you can select a number of bases up/down stream. If you want 1-- either side of your PCR product, select at least 150bp in this box, so that you have enough room to pick a nice primer. As to template, use genomic DNA, mouse of human depending on your system really. I prefer to use mouse (129/ola or C57 or FVB, I don't think it makes too much difference).
Another method could be to synthetize both strands of the pre-mir sequence complete of "sticky-ends" of interest, anneal them and clone. the problem here is that you have to find someone who sells long primers (which are also more error prone). I have only 30/35 extra bp flanking the pre miR sequence but it works very well. also tried PCR cloning but to me is much more time consuming (it is not always easy to find the right couple of primers in "difficoult" regions) and while giving no advantages in "mature miRNA yield"