pre-miRNA (ambion) transfection troubleshooting - I am getting opposite results than expected (Sep/12/2010 )

Hi guys,

I posted this under the molecular biology topic before, but now i am posting it here.

I am using the ambion pre-mirs to transfect cells to look at their effect on the endogenous levels of my target. I confirmed the target by luciferase assay using an artificial hairpin construct that i cloned which would express my mirna, but I wanted something that would have a flourescent tag so that I could monitor transfection effieciency. so i bought the pre-mirs from ambion with the Cy3 labeled negative control. I used 10 and 20 nM as my starting concentrations. Even the 10nM had a very high transfection efficiency, however when i blotted for my target protein, I actually observe the opposite effect...instead of decreasing it actually increased.

Do you guys have any suggestions as to why this is happening? any help is greatly appreciated. Do you think that the cells could be compensating and upregulatin the gene after i have knocked it down....should i try to use even less pre-miR?

First let me clarify that the so-called pre-miR from Ambion is not precursor miRNA but mature miRNA mimics.

Did you see protein downregulation with your "artificial construct"? What does your construct look like? What don't you construct and express pre-miR?

It is interesting to observe overexpression. Compensation is one possibility, translation activation has actually been reported. To get decent downregulation of protein, you may want to try a higher concentration ranging from 50-100 nM.

Make sure your Cy3 labelling actually gets into the cytosol. We had trouble with it just sticking to the cell membrane and thus giving false positive results for transfection efficiency. Apart from that what transfection method do you use for the Ambion product? Is it the same you used to transfect your plasmid? Some transfection methods cause problems like activating the cell and some gene expression.
Furthermore: did you use the same control sequence in your plasmid construct as you do with Ambions product? There are a lot of "control" sequences out there for RNAi which actually have a strong influence on the gene expression ... That than causes wrong read outs for the target gene/effect of course.
Make sure your control is a sequence that has been shown to be a true control instead of off target trigger, this seems to be the case for a lof of published GFP and luciferase targeting sequences. Scrambled sequences can let your cell go nuts, especially if they contain to have by coincidence immunoactive motifs and you have cells sensitive to this.

HI everybody,
Im very new to the miRNA field and I must say reading posts at this forum is more eye-opening than reading 5 papers on the relevant subject. For eg. I just learnt that pre-miRs from AMBION are actually not pre-miRs but mature miRNA mimics. So if I use this pre-miR, is it that these molecules would not be processed by the endogenous microprocessor complex and therefore yield a more physiologically relevant biological system to monitor? SO then, my question is what do we use for pre-miRs and from where??

Thanks for your suggestions or replies in advance
Swati

There are two strategies for producing precursor miRNA: the first or the easiest is cloning a pre-miRNA sequence into an expression vector and transfect the vector into your cells. Remember, to clone the pre-miRNA, you need to include 50-100 bp flanking seqeunce on each end. The 2nd method is to chemically synthesize the pre-miRNA (which will form a hairpin structure) and transfect the RNA into your cells as you do with siRNA.

How did you solved the problem with membrane labeling with the Cy3 labeled control pre-miR ? We are having the same problem. Cells incubated with the control, but not electroporated are becoming labeled

Thank you in advance.

wincel on Sat Sep 25 00:14:32 2010 said:

That is tricky but microscopy (confocal) helps to see what goes on. There is something called gymnosis though where cells take up naked small RNAs without electroporation too. This is mostly a matter of the concentration of the RNAs and the cell type and could be related to your problem rapane. What I was refering to is: it sticks on the surface only but thus gives false positive results of "incorporation".