I am aware that Pyro is the gold standard to validate a methyaltion study. However, it has restrictions like it can read up to 70-100 bp only. So, if I want to do Methylation specific PCR which is mainly nested PCR with specific primers , how good or bad is it in comparison to COBRA which uses Restriction Enzyme as well.
In MSP, you amplify the bisulfite converted DNA with two pairs of PCR primers (one pair specific for methylated DNA and the other for unmethylated DNA), which span the CpG site in question and qualitative results are obtained in the form of amplification of the sample by one of the primer sets. While in COBRA, the bisulfite converted DNA is amplified by a single set of primer and then restriction digestion of the sample is performed. The results in case of COBRA are both qualitative and quantitative. Hence, in my view, COBRA is preferable in sense of sensitivity and quantitation both.
Thank you Samriddhi,
In COBRA, I am exactly not sure how the quantitation is done. Is it by the formula
%methyaltion+100X(undigested/undigested+digested)?
or is it by electroblotting solely?
Shyamashree, This article might help you in quantitating your results.
http://onlinelibrary.wiley.com/doi/10.1002/elps.200900204/pdf
Thank you Yao Yang. Actually bisulfite sequencing might not be feasible in our lab.
Samriddhi, I can't find the attachment.
MSP is sensitive and best for detecting low levels of methylation, while COBRA is better at quantifying the percentage methylation.
Quantitative estimation with MSP is awkward and sensitive to standardisation. In essence, you're estimating methylation by the a ratio between two Ct value estimates. As these are measures of exponential amplification it gives inaccurate ratios and hence methylation estimates.
In a paper a few years back, I compared a PCR-based and digestion technique. The supplemental material has data on this. FIgures S6 and S7 are illustrative. http://www.biochemj.org/bj/432/bj4320323add.htm
Thank you Jason. You are correct that COBRA gives the percentage methylation. Actually it will be helpful to me to understand the exact concept of COBRA.
As far as I know it is similar to MSP but it employees RE and then based on the undigested control and digested band it compares the two.
But, I was wondering what all REs should I consider ?
Another method to consider is methylscreen--which does not require bisulfite conversion, but instead relies on the use of methylation sensitive and methylation dependent DNA cleaving enzymes. (see attached paper and link).
http://www.oriongenomics.com/technologies-clinical-assays.html
Of course, if you have access to a qPCR instrument with fluorescent detection, you could also consider high resolution melt analysis.
For COBRA analysis, the enzyme to use is the enzyme which shows, after bisulfite conversion, a different digestion pattern depending on whether the sequence was methylated or not. If you are looking at some frequently studied region, then probably a literature search will identify the location and the enzyme site to choose. If you are looking at a novel region, then you will need to perform an in silico analysis--convert the genomic sequences (CpG methylated vs. CpG unmethylated, all unmethylated C's -> T's) and then compare them to identify instances where one is cut by a restriction enzyme and the other is not.
you can use this software in silico:
http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi
http://www.ncbi.nlm.nih.gov/pubmed/12424112
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