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MSP: No PCR product for one cell line but yes for the other cell lines


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#1 mrpcr

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Posted 26 March 2009 - 10:20 PM

Hi,

I designed my MSP primers using Methylprimer. My problems are:

1. Some of the MSP and USP primers were able to pick a band for one cell line, said HT29 but not the other cell line, said Caco2. The positive control for MSP and USP was worked well in my every MSPCR run and the negative control remained clear.

2. Interestingly, the Caco2 that showed no amplicon for MSP and USP also showed negative expression for the studied gene. However, in HCT116, the same gene was being expressed even no amplicon was detected for MSP and USP.

Can anybody explain this. Your help is very much appreciated.

#2 epigenetics

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Posted 27 March 2009 - 07:42 AM

I also faced similar problem, but with patient samples. I had two patient's skin fibroblast with same disease. Got bands in one, not in other. One thing i thought about my DNA quality. But atleast according to Nanodrop calculation and gel, it was good.
Other might be i thought after my MSP, may be that patient has SNP at the region where primers are binding, not sure of this.
In this forum i got one answer that its possible that two patients has different C methylated, and as MSP primers are very specific, so it is not working.
So till now these are the answers i have. I also got suggestions that MSP gives artifact a lot, so BSP is much better than MSP. You can try it.

#3 pcrman

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Posted 31 March 2009 - 08:26 PM

If the DNA sequence is the same from the two cell lines, ie, no sequence deletion, no polymorphism that affects priming, then the problem is with the template -- DNA quality, yield after bisulfite modification, rate of conversion, etc.

If you are not screening a lot of clinical samples, I agree with epigenetics that you should try bisulfite sequencing method which gives you high resolution methylation mapping.

#4 mrpcr

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Posted 01 April 2009 - 09:01 PM

[quote name='pcrman' date='Apr 1 2009, 12:26 PM' post='20637']
If the DNA sequence is the same from the two cell lines, ie, no sequence deletion, no polymorphism that affects priming, then the problem is with the template -- DNA quality, yield after bisulfite modification, rate of conversion, etc.

Thank you very much for your reply. But I don't think the problem is come from the template because the Caco DNA is amplifiable with other MSP and USP primers. Maybe deletion can be the cause. When I screened with clinical samples, some of samples showed no detectable amplicons for both MSP and USP for that particular gene.




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