I also used the MethylFlash Methylated DNA Quantification Kit (Fluorometric) from Epigentek like Dr. Watson (Miousse et al. EMM 2013, and Miousse et al. RadRes 2014). It's pretty easy to use, I'm happy with it. The other approach that we've used it to do methylation-sensitive restriction digestion with McrBC followed by qPCR of the LINE1 repetitive element. This is very affordable, quick, and easy. Copies this element covers just so much of the genome that it is a proxy for whole-genome methylation status. In addition, it is also an indication of genome instability of it is hypomethylated. We use the two approaches in parallel most of the time.

I am afraid you will need sequencing for the"whole genome" methylation analysis. If you only look at CpG islands then HpaII can help to create library . Search for A. Bird papers from early 90ties.... BTW if you use HpaII you won't see anything on agarose unless you label with P32

The classical method - digestion of gDNA to single nucleotides and capillary gel electrophoresis. However one need to have access to apparatus. Another way - thin layer chromatography with a procedure similar to one described in this paper for 5hmC http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715015/.

I also used the MethylFlash Methylated DNA Quantification Kit (Fluorometric) from Epigentek like Dr. Watson (Miousse et al. EMM 2013, and Miousse et al. RadRes 2014). It's pretty easy to use, I'm happy with it. The other approach that we've used it to do methylation-sensitive restriction digestion with McrBC followed by qPCR of the LINE1 repetitive element. This is very affordable, quick, and easy. Copies this element covers just so much of the genome that it is a proxy for whole-genome methylation status. In addition, it is also an indication of genome instability of it is hypomethylated. We use the two approaches in parallel most of the time.

We have developed a LC-MS/MS method for global DNA methylation is suitable for you! haha~ It is selective, specific, very accuracy, fast and easy.^_^

http://pubs.rsc.org/en/Content/ArticleLanding/2012/AY/C2AY25140J

Hi Alicja,

The way you are mentioning is possible, but there needs to be a gold standard. I myself tried to perform such, but the measurement of the DNA smear went out of control. Instead, what I am doing now is to do MS-RAPD. It is rapid, minimum and cost effective. But does have some loopholes, since the primers are very very small. On the other hand you can perform a Southern using LINE-1 probes. I know it will not be cost effective, but I guess it will much more definite than going for NGS or whole genome sequencing.

Hope this helps.

Best.

Hi Alicja,

Mass spectrometry is the gold standard for global analysis of DNA modifications. If the changes in global meth are expected to be large you could try a dot blot where gDNA is spotted on nitrocellulose and probed with 5-mC (or the other modifications) antibodies followed by secondary ab (fluorescently labelled or HRP). Diagenode or Active Motif have the antibodies.

Best of luck,

Santiago.

I thought I'd chime in to suggest LUMA ( luminometric methylation assay). It is a bit more robust in the type of data it can give you when compared to immunoassays.

It uses MspI and HpaII (two methylation sensitive and insensitive restriction enzymes). These enzymes give you sticky ends amenable to pyrosequencing. The ratio of MspI and HpaII cutting ability from pyrosequencing gives you a very nice index of genomic methylation that is specific to CG context. It also uses an internal cutter, EcorI, to standardize MspI and HpaII cutting. The whole thing takes 2-3 hours for the digestion and a 20 minute run on the pyrosequencer (20-30$ a run for 24 samples).

The limitations are that it isn't comprehensive (it won't tell you where in the genome methylation changes) and bidirectional changes of equal amounts will cancel each other out. It's a great cursory technique to follow up on with more rigorous methods. I've used it in liver,muscle, brain, whole animals in a variety of model systems (ants, squirrels, mice, fish etc.)

Article Using LUMA: a Luminometric-based assay for global DNA-methylation

Article Application of the LUminometric Methylation Assay (LUMA) to ...

Another vote for LUMA (as mentioned by Sebastian G Alvarado above).

Fast, easy, reproducible, and if you see any differences then you go on the methyl-Seq, knowing that you have differences......

Hi Alicja,

There are several ways to detect the global methylation analysis:

1) Mass Spec.

2) ELISA

3) rtPCR

4) Southern Hybridization

5) RE based RAPD.

Each one of pretty reliable, based on the aspect you are looking for and subsequently discussing in your publication.

To me the whole genome sequencing is the best method, where you can utilize NGS platforms as well. ELISA givers you a good idea and so does ICC, where you can see the 5MetCyd, as it is a general protein that caps the mCs.

I would also suggest if you could look into the LINE methylation status using qRT-MSP PCR. It is also a good parameter.

Based on your study, you decide on the strategy.

Best,

Somnath.

How expensive is it to perform any of the techniques, above? Isn't it necessary to get whole genome sequence first?

The methylflash ELISA kit for 96 samples  from epigentek will cost you around $500-600. It will give you rough idea of % methylated cytosines

Hi Alicja Pawlak,

I suggest you to use MeDip-NGS for the identification of differentially Methylated region.

good luck.