I'm having a major issue with ISH of miRs. So I have FFPE mouse hearts which have been subjected to ischemia reperfusion. I've been at this a few weeks. So far I know that my positive control works (miR-1) but it does not work in my mouse tissue (it does work in human cardiac samples I have from another study). When doing my staining, U6 is working well in the mouse tissue, but miR-1 is not.
The tissue have been subjected to Evans blue staining and also TTC staining (for ischemic tissue) earlier in the study. Would these have an effect? How different is the preservation of the U6 from the mir-1 or other miRs present in the tissue. I have varied the proteinase K a little, but not extensively, would this have a signifiant impact on my tissue, considering U6 is working very well, and miR-1 is giving no signal.
Any advice is appreciated.
Hi Michael... what kind of probe and labelling are you using to detect miR-1 ?
DIG labelled LNAs to detect the miRs. Then antiDIG and AP afterwards.
OK.. you are using the most sensitive system that I know... Perhaps the Evans blue is preventing the access of the probe to the cells.
Is there a difference between murine miR-1 and human miR-1? This would explain why it would work in the human sample and not the mouse... Does the mouse tissue express this miRNA? U6 is very stable and conserved so one probe would probably work to detect U6 in both species...
There is homology between all species for this miR - the probe is essentially a 20 base LNA that binds to the miR-1 so species is not the issue really, but a good suggestion all the same. miR-1 is globally expressed in cardiac tissue, (in this case it is actually a miR control that I know is expressed in the tissue (and also mouse tissue) before i move on to miR-s of more interest to my project). I feel it is either a problem with evans blue/ttc interaction as suggested by Francisco, or maybe an issue of overfixation with 10% Formalin, although more suggestions still welcome
Hi Michael, I am currently trying to optimize miRNA-ISH using a suggested protocol by David Rimm. It however, uses the TSA plus Cyanine 5 signal amplification system as a method to detect the signal, so its fluorescence based as opposed to AP. I am currently using fixated cell pellets to do this, and similar to you I get a very specific and clean signal for U6. However, there is a lot of non-specific signal for the particular miRNA i am interested in. Although, this protocol uses a different system, it might be worth looking at the paper, perhaps modifying your initial processing steps might prove to be useful The PMID for the paper is 22482439.
Hi Michael, do you know ACD Advanced Cell Diagnostics (www.acdbio.com)? The probes are very sensitive and specific for many target mRNAs. It's possible use chromogenics and fluorescents in ISH reaction.
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