Hi all, I wonder how you measure miRNA in a sample retrieved from a tumor in a xenograft model. After tumor developed, miRNA was injected intratumorarly to look for effects on tumor growth. If you do qPCR on the sample (using specific primers) to assess the concentration of miRNA (to make sure miRNA is sufficiently in there), what will you be measuring: miRNA oligos (which seem impossible to amplify??), or the expression of miRNA gene. Maybe funny, but this is confusing to me! Another thing I'd like to understand, in the miRNA arrays, what company puts in the chips or the plates (like ones from SABiosciences) are genes (DNA) coding the miRNAs or the miRNA oligos?. Thanks for any input
That is a complicated question. You injected the precursor, double-standed RNA (mimic), plasmid DNA containing/expressing the microRNA?
I am not sure whether any of them would work to get the microRNA into your tumor cells and expressed well. If you injected the "microRNA" in form of a RNA, I think, it might get degraded before it gets into the cells, if it gets into the cells at all.
Therefore, my feeling is that the technique to express the microRNA in the formed tumor may not have worked.
Let's assume, it did work, then you should for a short period of time observe an increase in the expression of this microRNA by qRT-PCR. I have no idea how long this expression would last, but probably not very long.
Assuming that you want to test whether your microRNA of interest can influence the phenotype of the tumor once established, you may want to look into an inducible system and generate a tumor cell line that has a microRNA transgene that is silent as long as you don't induce it (perhaps a doxycycline/tetracycline inducible system?).
If you want to explore a therapeutic application and you already know that your microRNA will inhibit tumor growth, then you are confronted with the problems of delivering microRNAs in vivo. I have seen the use of inhibitors but not of the actual microRNA... That would be a task i itself, figuring out how to deliver successfully your microRNA of interest in sufficient amounts to the majority of tumor cells to alter their phenotype.
Not sure, I answered your question...
Hi Jahangir
First question: if you inject miRNA mimics to the tumor and then extract RNA to measure miRNA levels, you will detect the miRNA that you have injected if you use specific primers. qPCR techniques for miRNA detection are based on a specific protocol for cDNA synthesis that uses either a initial poly-adenylation step or a hairpin loop primer, and using these protocols you will detect the injected miRNA. Only if this miRNA mimic has some chemical modification (in 3' or 5') that prevents the RT reaction you will not detect it.
Second question: former SABiosciences (now Qiagen) low density arrays are basically PCR plates with specific DNA primers for each miRNA located in the wells. Each pair of primers in each well will amplify a particular miRNA. Some other manufacturers use different strategies, for example including modified oligos, but the idea is the same: to do a specific PCR amplification for a different miRNA in each well.
Hope it helps. Best
Paco
HI Thomas:
Many thanks for the excellent note. Actually we injected synthetic miRNA oligo (mimics) in a lipid-based formulation directly inside the (developed) tumors for a specific period of time. This miRNA proved to induce apoptosis in vitro. The confusing thing is that we saw increased expression of miRNA compared to control, I am still not sure (as I mentioned above) where this induced expression comes from???!!! (it was reproducible in all samples).
As Paco indicated: if you use a mimic, it should become detectable by the qPCR assay since the cells will make the mature microRNA out of the "mimic" (I assume a double stranded siRNA like RNA). Once that gets into the cells, they make the mature microRNA, hopefully incorporate into the RISC and start to inhibit certain genes (in a perfect world).
So, it is perfectly fine if you see increased expression of this microRNA since you "dumped" massive amounts of it into the tumor. Doesn't that make sense?
Dear Paco and Thomas:
Thanks for your explanations which are quite clear to understand. So this induced (increased) expression is actually due to the injected oligos (which absolutely makes sense) "not to the induction in expression of miRNA genes", right? (this is the point I am trying to grasp!!!, just how to be sure the miRNA is in there!!, the question is not on the expression but on existence).
Yes I believe you are correct, from my experience injecting miRNA mimics should not induce amplification or endogenous miRNA expression. siRNA in plants and nematodes has been shown to induce endogenous expression of secondary siRNAs, which can cause system wide siRNA silencing, but this isn't seen in vertebrates or with miRNAs.
Just make sure your primers detect mature miRNA, if they are detecting the precursor miRNA then the increase in expression may be due to endogenous changes after injection. You injected mimics, and these mimics should be mature miRNA that are around 22 nt long, and your primers will detect a specific miRNA in the cells. Since you injected miRNA mimic and mimic control, lets say miR-155, then if you do qPCR on both the mimic and control samples you should see a major increase in 'expression' of miR-155 in the mimic injected cells over control. This tells you that the change in expression was due to exogenous injection and not endogenous expression. The miR-155 you would see in the control injected cells would just show you the endogenous miRNA levels that are in the tumor cells.
I am just writing my MSc thesis now on miRNA regulation. Give this paper a read, it is the most thorough paper I have found that explains siRNAs and miRNAs and their differences:
Cell. 2009 Feb 20;136(4):642-55. doi: 10.1016/j.cell.2009.01.035.
Origins and Mechanisms of miRNAs and siRNAs.
Carthew RW1, Sontheimer EJ.
PubMed ID: 19239886
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