Usually after conducting Methylation specific PCR in order to validate the methylation status, one should sequence to see which cytosines are methylated.However if one does not have sequencing machinery, what is the cheapest and convenient technique one can use to analyze methylation specific PCR results?
Realtime PCR based HRM analysis can determine methylation level if you do not have sequencing capability. But, the PCR primers cannot overlap with CpG sites.
You can use Combined Bisulfite Restriction Analysis (or COBRA) approach. This technic allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. It is an old fashion technic, still used and cheap.
https://academic.oup.com/nar/article/25/12/2532/2901842
Depends on your research question, if you just want the qualitative answer (Yes/No methylation), then running the PCR products in agarose gel should be fine however with following considerations ->
1. MSP is done with two different primer pairs: one specific for methlyated allele, and the other to unmethylated allele
2. in addition you should run parallel PCR controls with 100% methylated DNA and non-methylated DNA.
Alternatively, CpG island microarray is more a global approach.
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