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Heterogeneous methylation in a same cell line


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#1 mrpcr

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Posted 11 March 2009 - 05:26 PM

Hi,

I have identified a few genes by using MCA-RDA (Methylation CpG Amplification - Representational Difference Analysis) method. When I checked the methylation status of these genes on cell lines (HT29, HCT116, Caco2) by using MSP, some of the genes shown to be both methylated and unmethylated on a same cell line. Can anybody explain this? For your information, I am using Qiagen DNA extraction kit and EZ DNA methylation Gold Kit (ZymoResearch).

Furthermore, some of the MSP primers can amplified for one cell line (exp HT29) but not the other (exp Caco2). How can I overcome this?

I would be grateful to any suggestion.

#2 pcrman

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Posted 12 March 2009 - 05:42 PM

I believe you are talking about partial methylation, ie, both USP and MSP primers can amplify products. This is quite normal and can be attributed to many factors such as allele-specific methylation, poor specificity of primers, DNA contamination, etc.

For your second question, do you mean both MSP and USP primers do not amplify one cell line? First make sure your template is good, check the region or the primer binding sites are not deleted, mutated in the cell line. If everything works, you should be able to see amplification at least from one set of primers (methylated or unmethylated).




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