Hello Kathirvel,

Easiest way to quantify MSP is to use qMSP as described here: http://www.bio-protocol.org/e871 or here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2648805/

If you don't have Taqman option, one can prepare control mixes of known 100% methylated and 0% methylated gDNA in different ratios:( 0.1% 1% 10% etc.) or prepare such samples from PCR fragments non methylated and PCR fragments 100% enzymatically methylated. Than you set up PCR with your controls and experimental samples and control mixes in LINEAR range. One would need to find linear range experimentally (Usually it's somewhere between 20-30 cycles). And than compare you samples signals with control mixes for semi-quantitative % level assessment,

I hope it helps. 

Hello Kathirvel,

Easiest way to quantify MSP is to use qMSP as described here: http://www.bio-protocol.org/e871 or here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2648805/

If you don't have Taqman option, one can prepare control mixes of known 100% methylated and 0% methylated gDNA in different ratios:( 0.1% 1% 10% etc.) or prepare such samples from PCR fragments non methylated and PCR fragments 100% enzymatically methylated. Than you set up PCR with your controls and experimental samples and control mixes in LINEAR range. One would need to find linear range experimentally (Usually it's somewhere between 20-30 cycles). And than compare you samples signals with control mixes for semi-quantitative % level assessment,

I hope it helps. 

Dear Kathirvel,

I agreed with Dmitry for using the qMSP instead of normal MSP. 

If you don't have option for qMSP, then go through with proper MSP controls for 100% and 0% methylation with spike in standard curve with 25%, 50% and 75% as well.

Further use proper bisulphite converseion control gene like b-actin and a housekeeping gene for loading correction.

for semiquantitatibve analysis you can use simply ImageJ software and calculate the band intensity as described nicely in below tutorial

https://www.youtube.com/watch?v=JlR5v-DsTds

and compare the intensities with the standard intensities to get the relative methylation percentage as in below example article:

http://www.ncbi.nlm.nih.gov/pubmed/22726460

Hope it helps and All the best

PS- irrespective of your question, for accurate CpG methylation, I would prefer the pyrosequencing to get exact methylation percentage.

Greets,

Tushar

Dear Kathirvel,

I partially agree with Dimitri and Tushar.

MSP with gel assessment (quantified by densitometry) is a really poor technique for methylation quantification, and you should clearly go with a qMSP at least.

However even qMSP has a major drawback which is possible amplification bias due to unequal efficiency of the primer binding/ and amplification between methylated and unmethylated template.

If you can try to use a methylation independent amplification technique such as HRM, or pyrosequencing, which will avoid amplification bias. 

Another important aspect of your experiment is your controls. Many are using mixes of artificially whole genome amplified DNA for 0% and MssI treated DNA for 100%, however you rarely achieve a complete demethylation and methylation, which can affect your experiments. 

Hope it helps,

Best,