MethyLight assay can give you a rough estimation of how much DNA methylation is involved for a couple of CpGs. It is better to use a multiplex assay with two different fluorescent labelled probes to do the real time PCR in order to minimize any variations. Probe design with similar property is critical too.

Pyrosequencing is a better assay to quantify DNA methylation for a short sequence (up to 200bp maybe) with CpGs. For a bigger CpG island, mass spect assay (Sequenom) is a better platform but it is tricky to use and not every CpG site is likely to be covered.

Hope this help.

You would have to use two differently fluorescence-labeled probes. That approach has also been described for quantifying the amount of unmethylated gene and methylated gene simultaneously in one run. In that case, you amplify the sequence in question with the same primer pair (with ideally no CpGs) and the probes differ in their ability to hybridize to their target sequence containing one or two (un)methylated CpGs. If you run a reference sequence (such as MyoD commonly used) - using of course different primers from your promoter of interest, you will have to make sure that the amplification efficiencies are indeed comparable.

Good luck

WAS

The problem of using this assay to quantify DNA methylation stems from the thermodynamics of probe binding. Even when using a reference (e.g. a DNA sequence that has no CpG sites), the real issue is that the probes (either for methylated or unmethylated strands) is not 100% specific. As you know, in PCR, one can use degenerated primers to amplify a sequence. This is possible because, even if a primer differs with one nucleotide from the priming sequence, it can still bind with some affinity, depending on where the degenerated nucleotide is. The same applies to the probe in the case of your assay. In other words, if there is a high degree of variability between adjacent CpG sites, the probe will bind with different degrees of affinity, and render a positive result. This is why the assay cannot discriminate between various combinations for the methylation of multiple CpG sites. It will give you an overall assessment, with a component of false positives.

I would suggest pyrosequencing instead.

MethyLight assay can give you a rough estimation of how much DNA methylation is involved for a couple of CpGs. It is better to use a multiplex assay with two different fluorescent labelled probes to do the real time PCR in order to minimize any variations. Probe design with similar property is critical too.

Pyrosequencing is a better assay to quantify DNA methylation for a short sequence (up to 200bp maybe) with CpGs. For a bigger CpG island, mass spect assay (Sequenom) is a better platform but it is tricky to use and not every CpG site is likely to be covered.

Hope this help.

I fully agree with the views of Dr. Ricky Yuet-Kin Leung

In our hand pyrosequencing is more reliable, as suggested by others.

Go with pyrosequencing, it worked for us and I highly suggest it for shorter sequences.

Pyrosequencing is mature method for your problem.

Thanks everyone for the valuable suggestion. I know pyrosequencing is more reliable to quantitate methylation but because of the available resources I have to stick to Methylight assay. I just want to know when we normalise the DNA input, how to use the normalised data for methylation quantification. I know it look very simple and basic but right now I am not being able to figure it out, so I need your help and some valuable suggestions.

I understand that you have to stick to your methods and use the equipment that you have (my students have to walk across the campus to use a pyrosequencing instrument...). So, if your sequence allows I would recommend that you try the approach with degenerate primers for U and M reactions, but differently labeled probes. This will give you a ratio that you can calibrate with a standard curve. That ratio is relatively independent of the amount of input DNA (as you should check for each assay), and you can use dilutions of your methylated and unmethylated standards to determine at which deltacTs the values become unreliable. I would like to give a reference for this method, but I cannot find it anymore. We have tried it several years ago (it works only of course, when you can find suitable primers for both M and U sequences), but then we switched to, well... pyrosequencing.

Best wishes

WAS

I suggest selecting a DNA sequence without CpG sites for your internal reference (dar away from the location of your querried sequence). Then use the delta-Ct-Ct method for data assessment. When it comes to statistics, because real time PCR data comes as ratios, you log2 normalize these ratios, then apply a parametric testing (should variances are equal) or a non-parametric testing if they are not. I would also like to mention that DNA methylation data is not necessarily normally distributed, as it ranges from 0 to 100% and, therefore, it follows what we call a beta-distribution. There is controversy on wether to apply parametric or non-parametric testing to such data. I would suggest applying a non-parametric testing first.

It seems from the producers web-page that the instrument you are using is capable of High-Resolution Melt analysis (HRM) I suggest using it for quantification of methylation. Here is an article that compares HRM to pyrosequencing: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052501

Here is more information on HRM: http://hrm.gene-quantification.info/

Not really sure, but this might be useful for you: http://www.ncbi.nlm.nih.gov/pubmed/20472822

Don't hesitate to contact me if you should have questions.

My suggestion is to use methyC-seq, or see whether you can refer the Science paper (341:1110, 2013)

We use pyrosequencing, a robust and reliable method for routine daily practice.

Dear Rahul, I would suggest to use Biotage pyrosequencing as a fully quantitative method for promoter methylation analysis and normal cellular counterpart of the tumor samples you are working with will probably be the best negative control.