quantitative methylation analysis? - (Jan/29/2008 )
Hi,
What is the best method to use if you want to quantify DNA methylation results? Is there a quantitative Bisulfite method or is q-PCR the way to go?
hey,
recently i come across something about methylation stuff,
u can use bisufate modification kit (time consuming)
1-DHPLC looks easier, only problem is u can't find out the methylation process at specific sites but perse u can quantify between two conditions (can say more or less quantitatively).
2- u can use methylation specific restriction enzymes, digest them, run on gel, more methylations sites more fragments..
gud luk
not sure where you are getting at hcmp
you may want to check out biq analyzer for bisulfite seqeuencing.
Nick
Hello again 
I have been studying the methylation pattern of the human genome. I have used methylation-sensitive and insensitive restriction enzymes - making it possible to generate fractions of methylated and unmethylated regions of the genome, done a PCR using primers specific for CpG islands of a particular chromosomal region, and run this on a gel. However, this seems like a quite old-fashioned way of doing it. Nowadays results vizualised on a gel are not enough. I would like to confirm my results using another method aswell but I don´t really know which one to use...... We have done a microarray experiment - but there seems to have been some problems with the interpretation, so I can´t really count on that at the moment... any ideas of what method to use?
bisulfite PCR and sequening is the gold standard and would be the one of choice for you!
good lcuk!
Nick
What is the best method to use if you want to quantify DNA methylation results? Is there a quantitative Bisulfite method or is q-PCR the way to go?
Hi guys, quantitative DNA methylation determination after bisulfite treatment: try pyrosequencing or MALDI-TOF MS --> for instance "Sequenom" Epityper. Epityper allows for high throughput analysis, pyrosequencing is slightly better but only 50-100 bp at a time....and not really high throughput...
I use Epityper and found 3% differences in MZ/DZ twin pairs that are reproducible (so not just because of variance due to bisulfite treatment...a big problem for quantitative determination of methylation).
hope it helps, bye bye
Bye ET2B,
P.S. I use EZ DNA methylation kit, without cycling, but ON 50 degrees bisulfite treatment --> best results
What is the best method to use if you want to quantify DNA methylation results? Is there a quantitative Bisulfite method or is q-PCR the way to go?
Hi guys, quantitative DNA methylation determination after bisulfite treatment: try pyrosequencing or MALDI-TOF MS --> for instance "Sequenom" Epityper. Epityper allows for high throughput analysis, pyrosequencing is slightly better but only 50-100 bp at a time....and not really high throughput...
I use Epityper and found 3% differences in MZ/DZ twin pairs that are reproducible (so not just because of variance due to bisulfite treatment...a big problem for quantitative determination of methylation).
hope it helps, bye bye
Bye ET2B,
P.S. I use EZ DNA methylation kit, without cycling, but ON 50 degrees bisulfite treatment --> best results
What is the best method to use if you want to quantify DNA methylation results? Is there a quantitative Bisulfite method or is q-PCR the way to go?
Hi guys, quantitative DNA methylation determination after bisulfite treatment: try pyrosequencing or MALDI-TOF MS --> for instance "Sequenom" Epityper. Epityper allows for high throughput analysis, pyrosequencing is slightly better but only 50-100 bp at a time....and not really high throughput...
I use Epityper and found 3% differences in MZ/DZ twin pairs that are reproducible (so not just because of variance due to bisulfite treatment...a big problem for quantitative determination of methylation).
hope it helps, bye bye
Bye ET2B,
P.S. I use EZ DNA methylation kit, without cycling, but ON 50 degrees bisulfite treatment --> best results
Are there places that will do the pyrosequencing for you if you are poor lab without the money to buy one of those fancy machines yourself. If so, does anyone have any recommendations on where to send your samples?