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McrBC + qPCR : mock has higher Cts than digested?

McrBC methylation

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#1 Vincent79

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Posted 30 April 2013 - 12:50 AM

Hi everyone, I performed McrBC restrictions on genomic DNA as well as a mock treatment (same mix and quantities, without enzyme only). I use roughly 10 units of McrBC per 100ng of gDNA. I then use approx. 5ng treated or mock DNA per qPCR reaction
Surprisingly, I observe that many of my mock samples have higher Cts compared to my digested samples. This is reproduced across several regions and experiments. This happens only for hypomethylated regions, when mock Cts should roughly equal treated Cts. The Ct difference is not high, usually up to 0.5-0.7 Cts, but is enough to bring negative enrichements [calculation used : 1-(1/2^delta Ct (McrBC digested – Mock treated)) * 100] .
I was wondering if anyone else has observed this kind of results, and if so how you dealt with it. I am wondering if the fragmentation of the treated gDNA enhance the efficiency of the qPCR allowing for slightly better amplification performance (if so, sonication may help?). Also, it could be error brought by the qPCR technology, but then it shouldn't be so reproductible. Finally, I observed that in some articles data wasn't quantified but delta Cts were simply treated as "if <1", "if >1",...) - is that because theMcrBC technique by itself cannot be used for a fine qPCR analyse..?
Any thoughts welcome.
Vincent

#2 pcrman

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Posted 07 May 2013 - 06:58 PM

I agree the difference in Cts is not that high, and can be regarded as normal variations without underlying difference in methylation status. Have you tried further diluting your DNA before the reaction? You expect to see increased methylation by the treatment? Are there positive control DNA available?




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