HPLC protocol for methylation quantification - (Nov/12/2005 )
Hello,everyone!This is my first post to the forum though i have take much notice of it for a long
time.I am doing the MSP,too.Not very smoothly,recently i met a problem!So i write to ask help!
My boss want me to quantify the methylation using the DHPLC.I even don't know what the DHPLC
is ,how could I apply it into experiment.Could someguys have the protocol of quantifying the
methylation using the DHPLC.Thank you in advance!
HPLC is high performance(pressure) liquid chromatography. It is a method of measuring products according to size and can separate all five nucleotides according to size. You basically digest your genomic DNA with a nuclease or something similar so the DNA becomes a soup of nucleotides. Run on HPLC and you get peaks according to size of the nucleotides the peak corresponds to the amount so it is quantitative as well.
what you can do then is measure global genomic DNA methylation by this method. What you can not do is localise where within the genome is indeed differentially methylated. So HPLC is great to measure differences in methylation between different cell types or tissues.
in terms of protocols, they can be found in early papers dating back to 1980's-1990's in NAR. I can't give you the pdf's because they were not made back then!!
just pubmed it and you will get some papers, Melanie Ehrlich's group was quite prolific in this area.
Hi methynick.Thank you for your fastreply!As what you have said ,DHPLC can quantify but not do
the quality of the methylation.I should digest the genomic DNA with a nuclease and run on
DHPLC!So i can not quantify the interesting sequence or product after the
MSP.Is that right?
precisely, gives you a good indication of a methylation difference between cell lines or tissues, you will have to perform RGLS or AIMS to then isolate the regions of differential methylation. However if you can detect a drastic change in methylation by HPLC, this makes it easier to find some regions that are differentially methylated with higher resolution techniques
I have been trying to analyse some samples with HPLC for a few weeks now. At present I am doing a lot of trouble shooting, but it is coming along gradually. I think my problems lie within the digestion of the genomic DNA. Most recent papers will reference back to a paper in 1980 titled, "Quantitative reversed-phase high performance liquid chromatograpic determination of major and minor modified deoxyribonucleosides in DNA", Nucleic Acids Research, Vol 8 Number 20. I think some of the HPLC methods they use are a little outdated, but it was written 20+ years ago.
The basic procedure is:
RNAse treatment with RNAse A and T1 to eliminate RNA that may influence your HPLC results.
Then digest first with DNAse I followed by nuclease P1. You may have some trouble tracking down the P1 enzyme as a lot of the papers I read reference companies which no longer supply it. I got mine from US Biological, and I believe Sigma may also be able to help. This enzyme cuts the DNA between each base, leaving you with mononucleotides. Following this alot of papers mention bacterial/calf intestinal alkaline phosphatase to de-phosphorylate, but I'm not sure this is 100% necessary. You should then be set to run your results on HPLC.
I am using a Supelco 25cm x 4.6mm 5um LC-18-DB column with Ammonium Phosphate buffer with methanol. I think there are a few different columns that you can use. Try the www.sigma-aldrich.com website and click the supelco link to see a range of columns.
I can give you more in-depth details if you wish, but that is the basic gist of it.
Davo: Is there a convenient set of standards for modified nucleotides? I'd like to look at the modified nucleotides in tRNAs, and there is a bewildering variety. Does someone make a mix for HPLC calibration?
I have a set of standards, "Nucleoside Test Mix", from Supelco that contain 12 analogs of the DNA bases in a few different forms. As I know little of modified nucleotides in tRNA, the composition of the standards are: sodium formate (as a preservative), pseudourdine, cytidine, 3-methylcytidine, uridine, 1-methyladenosine, 2-thiocytidine, 5-methylcytidine, 7-methylguanosine, inosine, guanosine, and ribothymidine. I think supelco may even have other standards which might be more suited for your needs, check their website (go via sigma-aldrich.com). I am regretting buying this mix of standards at present though. As I am trouble-shooting and trying to confirm the identity of some peaks, it would be easier for me to buy the standards individually, and eliminate some standards which are not in DNA! At present I don't know which companies can supply the individual standards, but I'm sure I've seen them around.
I have been using LC-MS method. You only need 10-100ng total DNA. Much more sensitive than HPLC and HPCE method. For the standard, I bought individual base from Sigma and made it up myself.
I assume the LC/MS method also uses nuclease P1 digestion etc? Could you please share your method regarding the DNA preparation? I use 50ug/ml of P1, however, when I run on HPLC I am getting about 10 peaks instead of 5, and I am thinking my digestion is not working 100%. I have attached a chromatogram showing the results following P1 treatment.
Denature your genomic DNA (8ul)100C for 3 minutes, the put on ice;
Add 2ul nuclease P1 (1 unit/ul from US biologicals) at 37C overnight (16hours);
Add 2.5ul 0.5M Tris (pH8) plus 1.5ul alkaline phosphatase ( 50units/ml from sigma) to the sample, 37C for 2hrs.
I got 5 peaks.