MeDIP DNA quantification - after MeDIP how can one quantify how much DNA is precipitated (Jul/31/2007 )
Hi. I just started working on MeDIP and I want to start Real Time PCR to validate the results. I don´t know very much about Real Time PCR but I know its important to know the exact ampunt of DNA one adds to the reaction.
So the problem is that the recovered portion of DNA after MeDIP is less than 1%. I start with 5ug and end up with about 50 ng in 60 uL. This is very small amount, in concentration and volume, to measure in a normal spectrophotometer.
1. How can I quantify efficiently the DNA after MeDIP so that I can measure methylation enrichment preciselly?
2. Can a standard curve on input DNA dilutions be used as a reference for all amplicons? (MeDIP is dependent on CG densitiy, so no 2 regions are picked up the same way!)
Thank you
hi there,
we use picogreen to quantitate the IP it's now called quantit.
quantitative real time PCR is used to determine if a known methylated promoter is indeed enriched by MeDIP, there are a number of promters we use and will depend on the cell line/tissue used.
Nick
we use picogreen to quantitate the IP it's now called quantit.
Thank you for the quick response. I have one more question. Picogreen is for double stranded DNA. When we do the MeDIP, we have to denature and then place on ice. Do you think that after all the IP procedure (including pheno/chlorophorm extraction), the DNA is in double stranded form again? I ask because we plan to buy this Qubit fluorometer (Invitrogen) for our lab, but it only measures dsDNA, like picogreen.
Thanks again :)
Diana
Diana,
you bring up a good point, I don't have concrete evidence that the IP remains single stranded throughout the whole process. My feeling is that yes you need to denature the DNA to expose the methylcytosine for antibody capture. As in most mammalian systems the methylation is symmetrical so theoretically both strands would be captured, after IP and DNA extraction I can't see why the strands won't reanneal.
Having said this, we get quantifiable results using picogreen on the IP fractions so there is something there after IP. We were tempted to buy a Qubit, but because we have a microtitre plate reader, we use this instead.
Nick