DNA extraction for methylation - DNA from cell-free serum for methylation treatment (Apr/22/2005 )
How to get the DNA from cell-free serum for methylation study with sodium bisulphite ?
My problem is that first, yield of DNA from 1 ml cell-free serum is very low, so that we can not get any product after methylation specific PCR, but it works very well for DNA from biopsy or tissue. We try to use several DNA extraction kits (Qiagen, Promega, Roch et al.) but it does not help.
The second problem is that the DNA from lymphocyte contaminated serum will produce positive bind for tumor suppressor genes (TSG) after MSP. Apperently it is a fulse result because the TSG shound not be methylated in norma tissue. Please give your suggestion. See the paper: Clin.Cancer Res, ´8, 1761-1766, 2002 by Lee, T.L et al.for your ref.
I didn't know cell-free serum contains DNA. If it does where is it coming from?
I know people have studied methylation of serum samples. The DNA in serum is leaked from necrotic cells. I don't know how much DNA you can get from serum samples but I know even pico grams of DNA is sufficient for methylation study. Theoretically, the minimum amount of DNA that works is the amount of DNA from one cell. I have used DNA from microdissected samples (containing dozens of cells) for bisulfite sequencing. During DNA recovery, I use glycogen as a carrier, precipitate DNA at -20C longer than usual, centrifuge for 30 min.
You can run a genomic PCR using untreated DNA from serum, if you can amplify something, then the DNA should be enough for modification.
If you are doing MSP, use nested primers, with outer universal primers (no CpG in primer) and inner M and U primers. You will need more cycles than tissue DNA.
If you are doing BSP, use either nested primers or run two rounds of PCR with the same primers.
Wow you learn something new everyday pcrman :-)
If small gDNA for conversion is an issue, try and get your hands on MethylEasy Kit from human genetic signatures just google them.
The kit converts DNA with no loss of it during the process. So you start off with 10 ng you end up with 10ng! It's a great kit and you can perform the conversion in four hours!
Does anyone have a good protocol for extracting DNA from microdissected tissue from parrafin embeded tissues?
My final aim is to do MSP on these DNA.
Please help as my supervisor and I are both new to these methods.