question about MSP bands - (Jul/30/2008 )

Hi!!

I'm doing MSP for different genes in different cell lines. In some cases, I observe a band when I use primers for unmethylated sequence and, in others, band when I use primers for methylated sequence. My question is... what happens when there are bands in both, primers for unmethylated and methylated sequence??? is the gene methylated or not??
Thank you for your help!!

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It is very common in tissues, you can see both methylated and unmethylated bands. Generally, it will be considered for a gene to be methylated if both are shown. That is reason why it is better to run qMSP or sequencing to quantify what percentage of methylation occurs

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Could it happen that bisulphite modification had been carried out partially??

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absolutely, and it could be that your pcr conditions are not optimal.

If these are ruled out, it is still possible, if you are say looking at a tumour biopsy for example, there could be a mixture of tumor and normal tissue giving rise to both methylated and unmethylated signals!

Nick

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and how can I know if pcr conditions are optimal or not??

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Based on my experience in qMSP used to test tumor tissues, we found methylation level for a particular gene varied widely.

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Hi larryking,
in my case, I've been using gDNA from different cell lines, so I would expect a high homogenity in methylation... Maybe I should carry out PCR in a higher restrictive conditions.

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I'm having MSPs where both the methylated & unmethylated reactions end up positive, as well. I consider it quite normal, since even in a cell line the cell population may not 100% homogenous in terms of methylation status. The problem gets even more complicated, I think, when the primers incorporate more than one CpG. In this case you won't be able to "see" the whole population with two reactions.

Godd luck..

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