Protocol Online logo
Top : Forum Archives: : DNA Methylation, Histone and Chromatin Study

QPCR with MSP - (May/16/2007 )

Hi, i'm just new in methylation field and i have a small problem with my qpcr.
I just run a qpcr with several bisulfite-treated DNA and genomic DNA treated with SSi as a positive control.
Even, i have a nice dissociation curve in all samples when i used my house keemping gene ( beta actin), and when i used Genomic DNA treated with SSI, but for the others Bisulfite-treated DNA i have several peaks. Is it normal?
Thanks for your answer...

-ptitced-

Hi, yes, it is normal. MSP picks fragments methylated or unmethylated at the primer binding sites. The sequence of the amplicon can contain both CpG C's or T's, depending on the methylation. So you have depending on the amount of CpG's within your amplicon a high sequence variability, which esplains the dissociations curves (I dropped that in my MSP protocols). In the fully methylated template, you should have a nice curves, as all CpG-C's should be prevented from modification.
But why do you use beta actin?

-krümelmonster-

Thanks krumel for your answer..
I choose beta actin as a hoosekeeping gene as they use in a paper. http://www.jbc.org/cgi/content/full/281/23/15763
Regarding the analysis of the result, given the fact there are several peaks, am i allowed to use CT value?
Thanks again for your help

QUOTE (krümelmonster @ May 16 2007, 07:09 AM)
Hi, yes, it is normal. MSP picks fragments methylated or unmethylated at the primer binding sites. The sequence of the amplicon can contain both CpG C's or T's, depending on the methylation. So you have depending on the amount of CpG's within your amplicon a high sequence variability, which esplains the dissociations curves (I dropped that in my MSP protocols). In the fully methylated template, you should have a nice curves, as all CpG-C's should be prevented from modification.
But why do you use beta actin?

-ptitced-

As long as you are aware that MSP is only a rough measure, you can. I would think about a series of mixing experiments to somewhat calibrate your measurements.

-krümelmonster-