MethyLight is probably the gold-standard to quantitate methylation using QPCR. This method employs fluorescent probes (TaqMan or similar), but depending on the region that you want to study, SYBR-Green detection might be also a good option. In many promoters, methylation generally occurs in an all-or-nothing fashion, so the methylated-target primers are enough to discriminate and quantitate the methylation levels (basically a regular MSP but using QPCR). As a reference amplification reaction, ALU reaction works very well in our hands.

MethyLight

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC102836/

MethyLIght on repetitive elements (conditions for the ALU reference reaction)

http://nar.oxfordjournals.org/content/33/21/6823

Best,

Sergio

MethyLight is probably the gold-standard to quantitate methylation using QPCR. This method employs fluorescent probes (TaqMan or similar), but depending on the region that you want to study, SYBR-Green detection might be also a good option. In many promoters, methylation generally occurs in an all-or-nothing fashion, so the methylated-target primers are enough to discriminate and quantitate the methylation levels (basically a regular MSP but using QPCR). As a reference amplification reaction, ALU reaction works very well in our hands.

MethyLight

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC102836/

MethyLIght on repetitive elements (conditions for the ALU reference reaction)

http://nar.oxfordjournals.org/content/33/21/6823

Best,

Sergio

I would recommend pyrosequencing for a more quantitative analysis of methylation patterns/levels at individual 'CpG' residues within your target sequences.

http://www.qiagen.com/Products/Catalog/Automated-Solutions/Pyrosequencing/PyroMark-Q24

If you have PCR instrument equipped with high resolution melting, the best method could be MS-HRM. It is very sensitive. You can detect methylation at levels as low as 0.1%.

http://www.ncbi.nlm.nih.gov/pubmed/17289753

Good luck

Pavel

MethyLight is probably the most used but also has disadvantage because it depends on methylation specific PCR and may introduce a bias.

Another method that wasn't discussed here is qPCR amplification of DNA digested by Methylation Specific Restriction Enzymes (MSRE) or in short MSRE-qPCR. We used to do it quite a lot and it was very useful as an quick, non-expensive, easy method to quantitate DNA methylation.

Now there are suitable kits like the qMethyl kits from Zymo-Research (http://www.zymoresearch.com/epigenetics/dna-methylation/region-specific-dna-methylation-analysis/onestep-qmethyl-kit) that could make easier still.

Having said that, we also have a lab pyrosequencer and I would say pyrosequencing is still the most suitable for accurate, publishable results of quantitative DNA methylation.

We used the Epitect assays from Qiagen, Like the answer from Gidon this method uses methylation specific restriction enzymes. There are existing primers for many promotor regions of if there was anything not already covered you can have custom assays designed for you. We had very reliable, repeatable results using this assay.

I am partial to MethyLight, as I have worked with this technology for over a decade. MethyLight is very good at discriminating DNA methylation differences between tumor and normal DNAs. As stated above, the use of the TaqMan real-time technology allows for quantitative data. MethyLight reactions are designed such that every CpG covered by the primers and probe need to be concordantly methylated in order to amplify, so essentially the assay quantitates the proportion of molecules in the sample with this one DNA methylation profile. Any heterogeneous DNA methylation will not be identified by the assay.

@Daniel.

Interesting remark, because the MSRE-qPCR is pcesicely the opposite.

It will digest DNA with any unmethylated CpG site and only molecules with methylated CpGs on all sites will be protected from digestion. Therefore, it quantitates the proportion of fully methylated molecules compared to unmeth and partially methylated.

Why not just use MeDIP (methylated DNA immunoprecipitation)? You can check Weber's paper on Nature Genetics 2006 for details. This method has been widely used and quite reliable.

We publshed a paper last year, it may be useful to you.

Quantitative methylation analysis reveals gender and age differences in p16INK4a hypermethylation in hepatitis B virus-related hepatocellular carcinoma.

Wang Y, Cheng J, Xu C, Liu S, Jiang S, Xu Q, Chen X, Zhuang H, Lu F.

Liver Int. 2012 Mar;32(3):420-8. doi: 10.1111/j.1478-3231.2011.02696.x. Epub 2011 Dec 23.

one key issue making your decision ...... depending on the source of DNA either native tissue or FFPE, I would suggest either MSREqPCR or qMSP (or variant formats), respectively.... easiest would be to ask people having the method running and send your samples :-) .... MSRE is simpler and faster, needs less optimizations,

....we use MSRE-qPCR in a high throughut format (on a Fluidigm Biomark) analysing 96samples x 96 assays....and have about 600 assays in the fridge, but have also long experiences with MSP & qMSP.... on FFPE however even using the bisulfite based approach, quantification is challenging as long as the pathologists do not really standardize or change to other fixatives than formalin....have seen dCTs > 10-15 for control genes comparing native vs FFPE, meaning that Ct values of control genes are at Ct >30-35, thus reliablility is very questionable in such situations....but that's another story

If you reallly want to use the realtime PCR i would use Quantification by taqman subsequent to DNA digest with a methylation sensitive enzime ike BSTUI, HPAII, HHAI. But to really quantify DNA methylation i would use pyroseq or even cheaper and covering more cpGs MassARRAY

This is a surprisngly difficult question and one with which we are constantly grappling despite considerable expertise in this area.

SMART-MSP is a useful alternative to MethyLight if you have access to HRM.

MS-HRM works very well but quantitation is relatively difficult for those many loci in which methylation is heterogeneous. We have reviewed the issues related to quantitation of such loci and this is open access like many of our articles.

if we have a cpg island of 800bp, can I use  QMSP (SYBR-GREEN or taqman) for testing? 

i have tried to get taqman probes for methylight reaction from ABI they are not providing BHQ quencher so can anyone tell me fron where i can get the bhq probes in india.

I have a question; I'm trying to use MSRE digestion and RTqPCR to detect promoter methylation, and we have both HpaII and Msp1 REs in our lab that I had obtained for in vitro plasmid methylation experiment. I've looked at a few papers using this technique, but  none of the ones I've came across have used Msp1 as a control.  Is it acceptable to just use an undigested sample and HpaII digested sample and not include an Msp1 digested sample?

I have a question, I did qMSP of methylated DNA and i got two melt curve.when i run that in gel two bands are amplifying one for my target band and another for primer dimer. Is this a problem in my qMSP experiment.

Neha Singh either you are using too low concentration of template DNA or you're using too high concentration of primers. Moreover, the annealing temperature may be inappropriate.