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Quantuitative staining of RNA and DNA?

DNA staining RNA staining mRNA quantification

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#1 Peterlorre

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Posted 04 September 2014 - 01:22 PM

Hello All, 

 

My lab has been working on a cell system in which we believe that the total per-cell mRNA production may be changing globally between conditions. I'm somewhat new to cytometry and I am wondering if there are established, hopefully quantitative methods for estimating the per-cell mRNA content, preferably in parallel with DNA abundance. I have looked around in the literature a little bit, and the two options that I have run across are a combined Hoechst/Pyronin Y stain, and acridine orange staining.

 

As I understand it, Pyronin Y mostly stains double-stranded RNA, and the measured abundance is mostly associated with rRNA. Acridine orange binds single and double-stranded nucleic acid templates, but apparently the stain requires an acid environment that denatures rRNA and mRNA, so their signal isn't distinguishable. I think that this suggests that one might be able to compare the RNA/DNA ratios for both stain conditions to get something like a population-level mRNA abundance, but it's not clear to me if this would work or if it would be possible to calibrate the comparison properly.

 

Or maybe there is something else that is obvious and everyone does all of the time? Maybe an antibody that only binds to capped mRNA molecules? The other obvious approach is to do a double RNA/DNA extraction to try and quantify things that way, which I am doing, but I'd like to have an orthogonal way to validate those findings.  Any insight that you all might have would be much appreciated. 



#2 Rsm

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Posted 11 September 2014 - 07:10 AM

Another idea would be a FISH against polyA, such as oligo (dT) coupled to FITC. I would recommend LNA probes for that... At least you can distinguish rRNA from mRNA.


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#3 Peterlorre

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Posted 11 September 2014 - 12:32 PM

That's an interesting idea; I haven't done any FISH- should I be concerned that an oligo (dT) probe would bind genomic regions as well? I assume that there is a denaturing step of some kind, but I'm not familiar with the details of the technique. 



#4 Rsm

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Posted 12 September 2014 - 03:11 AM

Well, the obvious control would be using a sense probe, like oligo (dA) LNA conjugated to a different colour (Cy3). This would bind with the same strength to genomic polyA regions as the oligo (dT).

 

Here's a protocol: http://www.ncbi.nlm....pubmed/19393610


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#5 Peterlorre

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Posted 12 September 2014 - 08:53 AM

Awesome! This looks really great. Thanks for your help. 






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