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#1 kerenshi



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Posted 08 February 2009 - 12:29 AM

I try to find out the correlation between expression and methylation status of new genes

I already found the promotor region and 2 CpG island near the initiation signal

I plan to find out the methylation status by MSP in tumors ves. normal tissue.
I want to plan the primers:
to my knowlage the methylation is not 100% - not every CpG will be methylated even if the CpG island will be shut by methylation
2. the primers contains only 1-2 CpG and those CpG could be the one that are not methylated!! - I feel I need more luck?!!
3. so - if I get positive result - thats mean - methylation. if I get no methylation - maybe I chacked the CpG that specifically does not methylated even though other CpG IN THE islad - are methylated....

I hope that I make my self clear cause I feel a little bit comfused......


#2 pcrman



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Posted 08 February 2009 - 11:16 AM

Hi kerenshi,

I know your purpose is to screen many tissue samples for methylation. ideally, you should first do a detailed mapping using bisulfite DNA sequencing on a few samples or cell lines so that you will know which CpG sites are important to transcription. Once you have that information, then you can design MSP primers on those particular CpGs.

Yes there is a dilemma when it comes to decide whether to have one or more than one CpGs in the MSP primers. if one CpG is in a primer (of course at the very 3' end), the primer will give very good specificity. If it covers two CpGs and they are not equally methylated, how the primer behaves is hard to predict. On the other hand, it all the CpGs covered by one primer are equally methylated or unmethylated, then covering more than one CpG will have better specificity than just covering one. Personally, i prefer to just having one CpG in a primer, and prefer bisulfite sequencing to MSP.

Another big problem with MSP is false positive/negative.

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