Global methylation assay - kit? - (Jun/01/2010 )

Hi,

I am thinking of using Sigma's "Imprint Methylated DNA quantification" to look for global methylation levels between my samples. Has anyone on this forum tested this? Does anyone have suggestions about this kit? If someone has, which method is more reliable, the single point assay or the standard curve method?

Thanks a lot!

-epicrazy-

epicrazy on Jun 1 2010, 07:35 PM said:

and? have you tried it or hit upon a better one?

-Dr. J-

I started with Sigma's kit -- it worked okay but I had a few problems with the color development step. I switched to the Supersense kit and it worked quite well and its fluorescence reader based. I did the standard curve method for both kits.

-epigenius-

Thanks for the info! Any tips for the sigma kit (in the colour dev step?), i already bought it and doing the assay today ? THanks in advance?

-epicrazy-

epicrazy on Jun 18 2010, 04:50 AM said:

Thanks for the info! Any tips for the sigma kit (in the colour dev step?), i already bought it and doing the assay today ? THanks in advance?

For me, I had to shorten the color development time because the reaction was very strong and fast, so keep an eye out for that. I had also increased washing time.

-epigenius-

Thanks for the suggestions! I tried it as well. It worked ok, not great. I had inter-well variability for technical replicates which was not good! Also, I tried it twice, first time the colour development was strong and the second time it was much slower. I could still detect differences between my samples (but my n was high, so i could get rid of the outliers). You have had no problems at all with the epigentek kit?

-epicrazy-

Hi epigenius,

I was wondering if you could give me a suggestion regarding calculation of methylation values from the kit. I used a standard curve method and plotted on a log scale, after which I calculated the amount of methylated dna for my samples based on the standard curve and converted it to real input. When I look for significance between sample groups, is the raw data my sample set or it it the converted input values? Since small variations in the absorbance can mean different input dna, I used a log scale. Any thoughts?

Thanks in advance!

-epicrazy-

Hi,

In my work I applied the Imprint Methylated DNA Quantification Kit from Sigma, in DNA samples from rat colon and colon mucosa.
Any of you had done the same? Or have knowledge of someone that already applied this kit in this kind of samples?

Thanks in advance!!

-Ana Pereira-