I am writing a project to study DNA methylation during infection and I want to measure global methylation taking advantage of a cell cytometer equipment.
I've been working on DNA methylation problem long ago. In that era global methylation were measured by other methods, using special HPLC approach, if I'm not mistaken. But I don't read anything about cytometer equipment used for this purposes. But that was long time ago (circa 2008)
you can do it.. check this paper here:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847606/
However, I`m not sure wheather flow cytometry is enough sensitive to give you a quantitative evaluation of DNA methylation. It depends a lot on the source of your DNA sample. And having worked with it I think you might have many problems in terms of reproducibility.
Vimentin (the molecule described in the paper) might be more expensive than doing a specific methylation PCR.
More sensitive and advanced techniques include bisulfite pyrosequencing, bisulfite mass-array (Sequenom EpiTYPER), bisulfite sequencing (if you have candidate genes those are the best you can use). If you are looking for genome-wide approaches you might consider the 450K Illumina beadcheap, or sequenced-based methods such as RRBS, MeDIpSeq.
If you give me some more info about the samples and the approach I might give you more precise information.
I`ve just read "global DNA methylation".. sorry... I can add this: you can use a method called LUMA that works on a pyrosequencer. This will be more reliable and cheaper.
Here`s teh paper:
http://www.ncbi.nlm.nih.gov/pubmed/21913077
Hi, I was planning to the the flow cytometry analysis of the DNA methylation. The method I was thinking about was based on the Immuno-staining of analysed cells with a monoclonal anti-5MeC then staining with FITC conjugated rabbit anti-mouse secondary antibody and analysed in flow cytometry.
There are some papers on this subject:
http://pubs.rsc.org/en/Content/ArticleLanding/2011/AN/c0an00790k
http://respiratory-research.com/content/13/1/74/abstract
Because I'am working on the tumor samples form the patients and I am not using the cultured cells, I currently use the MS-PRC or BS-PCR methods, or mentioned above HPLC istead of flow cytometry.
I just want to add a comment to the post of Karolina. MS-PCR and BS-PCR are suitable methods when you expect to see relatively huge methylation changes (such as the ones in cancer)..when you want to see smaller differences they are not enough sensitive.
I forgot to mention there`s another method appliable to a rela time PCR, called MethyLight, published from Peter Laird. This is also a quantitative method and very sensitive.
You might want to go for it if you have at least a real time machine.
Dear All, How do I measure Cytosine to Thymine / Cytosine to Uracil mutation bias?
Given the rate of spontaneous deamination, that results in C-> T mutation if C is methylated or C->U mutation if C is not methylated.
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