Real-time PCR to analyze DNA methylation - (May/01/2007 )

Hi,
I have to analyze DNA methylation using Real-time PCR. The strategy is to digest the genomic DNA using methylation-sensitive restriction enzyme and PCR amplification using specific primers. Methylated site will prevent the cut and therefore will be amplified; on the other hand, unmethylated DNA will not. So the quantitative analysis of PCR product will allow to determine the methylation status at specific sites.
I have never done real-time PCR, and nobody in my lab have used this technique to analyze DNA methylation.
I hope some researcher of this group might have experience in such analysis. Please give some idea in this regard, it would be a great help.
I will use Roche’s Light cycler for this.

I got this strategy from- The Plant Cell, 14, 857-867 (2002), Curr. Biol. 11, 747-757 (2001).

Thanks in advance.
dnanalyzer

Hi,

you'll probably find advice in the DNA methylation forum. I wouldn't use digestion + real-time, as you get very funny results depending on the quality of the enzymes (we used it, believe me...) + you can only analyze one site at a time (which may not represent the whole promoter). I'd prefer bisulfite based techniques, which can also be used with real-time (MSP).

Krümel

Hi Krumel,
I was in hurry to finish the DNA methylation analyis to include it in my PhD thesis, that’s why I choose this enzyme-digestion+RealTimePCR strategy. I have done it for few samples, but the result seems not convincing. I need to think otherwise….some other quick strategy………………..
Thanks for your suggestion.

dnanalyzer

Try bisulfite (with a kit, like Epitect or Methyleasy) and than do real-time MSP. That is fast and more accurate than the digestion.