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DNA Enrichment for Methylation Analysis


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8 replies to this topic

#1 Muhammad Umer

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Posted 25 August 2009 - 11:03 AM

Hello all
I am doing my research on methylation profiling of various TSGs in hepatocellular carcinoma. The sample for me are the fine needle liver biopsies. These I guess are a little small and the DNA which is expected to come out of these might not be more than 2-3 ug. I have a little larger set of TSGs to profile and also there is the issue of repeat experiments etc so I want to increase the quantity of my DNA.
The point is that I want to include a DNA enrichment step in my work. I have a couple of protocols with me like "whole genome amplification". I want the experts here in this forum to comment on this method and also please let me know if there is any other technique I can employ to have larger quantity of DNA.

Thanks

#2 Muhammad Umer

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Posted 28 August 2009 - 02:10 AM

nobody knows about this topic?

#3 methylnick

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Posted 01 September 2009 - 07:46 PM

sorry, been on conference leave :P

when you mean whole genome amplification, do you mean WGA on bisulfite converted DNA? or a MeDIP then WGA?

NIck

#4 Muhammad Umer

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Posted 01 September 2009 - 08:01 PM

well i mean WGA on bisulfite treated DNA. you can also look at these files im attaching

Thanks

#5 methylnick

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Posted 05 September 2009 - 01:50 PM

qiagen has a whole bisulitome amplification kit.

with such kits you need to test in your own hands to ensure there is no bias between methylated and unmethylated templats by comparing a control region before and after amplification!!!!

I have tried it and for our control region it seems to work fine, but I am always skeptical with the test regions we have looked at.

good luck

#6 Loretta

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Posted 20 October 2009 - 12:16 AM

qiagen has a whole bisulitome amplification kit.

with such kits you need to test in your own hands to ensure there is no bias between methylated and unmethylated templats by comparing a control region before and after amplification!!!!

I have tried it and for our control region it seems to work fine, but I am always skeptical with the test regions we have looked at.

good luck


Hello, I am new in the forum and I have exactly the same problem with my sample, since its DNA concentration is far below the 1 ug recommended for bisulfite treatment (I use CpGenome from Chemicon). I started to consider the possibility of using a kit to amplify the DNA already treated, like the whole bisulfitome amplification kit from Qiagen. Is there another opinion regarding this issue? And by the way, this question goes to methylnick, what kind of control region do you use to test a proper amplification? Please HELP ME!!
Loretta. :lol:

#7 racingstud

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Posted 22 October 2009 - 01:52 AM

Hello, I am new in the forum and I have exactly the same problem with my sample, since its DNA concentration is far below the 1 ug recommended for bisulfite treatment (I use CpGenome from Chemicon). I started to consider the possibility of using a kit to amplify the DNA already treated, like the whole bisulfitome amplification kit from Qiagen. Is there another opinion regarding this issue? And by the way, this question goes to methylnick, what kind of control region do you use to test a proper amplification? Please HELP ME!!
Loretta.


I've used the Applied Biosystems MethylSeqr conversion kit with some success. It only requires 300ng of DNA input so it might be an alternative to WGA.
As far as control region I am interested in what methylnick has to say as well.
Good luck.

#8 methylnick

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Posted 25 October 2009 - 02:46 PM

what kind of control region do you use to test a proper amplification? Please HELP ME!!


Anything that you know works in the past for you, we have plenty of assays that we know work in our hands, are you able to source some assays from another lab already working on this stuff? I know with the HGS methyleasy kit there are primers included in the kit to test for conversion.

In terms of amplification bias post WGA, I would reccommend a known imprinted region where you would expect approximately 50% methylated to unmethylated alleles in your sample, any significant biases will skew to the methylated or unmethylated allele accordingly.

Good Luck!

#9 NXT-Dx

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Posted 22 May 2012 - 07:08 AM

Hi

We have set up a company that offers methylation sequencing services and have processed over 1500 full methylomes and only need 500ng of DNA per sample (for the profiling service).

We have 3 types of services:

1) Genome-wide methylation sequencing (profiling)
2) Bisulphite sequencing
3) mRNA, full RNA or directiona RNA sequencing

We are always open to working with new people (service or collaboration-mode).

Maarten

www.nxt-dx.com




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