Whole Genome Amplification of bisulfite treated DNA - (Aug/31/2010 )

Hi, I just read the discussion about WGA of bisulfite treated DNA hold last September. Are there any new comments about which method is the best (Multilpe Displacement Amplification like the Qiagen Kit) or the Primer extension preamplification method? Does anyone has experience with the OmniPlex Method (eg. Sigma Kit) which generates a fragment library before amplification?

To check on the reliability one can look at imprinted genes in the amplified DNA, how many genes do you check?

Thanks, Rudi

Can't say I have tried the sigma kit,

as well as an imprinted region as a QC sanity check, I would also perform an unmethylated loci such as GAPDH and a methylated loci such as DDX4 to ensure there are no biases as both ends of the methylation spectrum as well.

good luck!

Thanks again for your help! I will try the quality checks!
Greetings Rudi

methylnick on Tue Aug 31 19:50:02 2010 said:

Hi Nick,

another question came up today, while thinking about my QC check. Is DDX4 methylated on both alleles in healthy humans? (I am working with umbilical cord blood of healthy termborn babys).

Thanks so far, Rudi.

that i am not too sure, I know DDX4 is a stem cell marker, you would need to test it yourself or find a paper that has already done this.

For most tissues I would say yes, and with cordblood, I would be leaning the same way but it's worth you checking for sure!

good luck

I've been using the Qiagen WGA Kit this week with puzzling results. Following amplification I am getting `900 ng/ul for my samples. However, when I try to perform BSP using the same parameters that I have used on un-amplified DNA I get no products. I have tried this on a couple of promoters where BSP has worked really well in the past with no success. Does any one else have experience with this kit?

ky_kev on Wed Feb 9 04:13:32 2011 said:

Hi Ky_Kev,

may I know how you quantify the DNA after the amplification? is it by spec?

I just started using the kit too. I'm puzzled by the fact that I'm getting ~1000 ng/ul (so that's similar to what you're getting). I started with 50 ng of bisulfite converted DNA and the Qiagen handbook said with that amount I should expect to get about 1000-3000 ng DNA yield - so with that I was expecting the concentration should be around 25-75 ng/ul.. and what I got is way way more than that. It should be a good thing to get more, but since it's too far off, I don't know what to make of my result.. since you're getting a similar concentration with the same kit, maybe you can share your thoughts on that?

Anyone else can help with answers maybe?

Thanks a lot!

fnad

fnad on Thu Feb 10 00:22:39 2011 said:

Sure, I used the ssDNA option on the nanodrop. The kit recommends Picogreen quantification but I didn't have access to that. What was your method of quantitation? I have since tried other BSP primer pairs and have managed to amplify from the WGA dna....so I guess my original primers that haven't been working need further optimisation. I am still puzzled as to why I got such concentrated DNA. FYI I have been diluting DNA down to 10ng/rxn.