RT-PCR or disulfite treatment, which is better? - (Mar/27/2008 )
Can someone please tell me which is the easiest and cheapest method to determine DNA methylation of plant gDNA? Can I digest gDNA with methylation sensitive restriction enzymes and PCR it? I am completely new to this. Thanks for any help.
yes you can digest genomic DNA with methylation sensitive enzymes, the most used (i think) are Hpa II and MspI, however there are others.
bisulfite conversion can be more complicated than rt-pcr, and can be used for different purposes. bisulfite tells you if your DNA is methylated by converting non-methylated cytosines into uracils, rt-pcr detects if the sequence you're analyzing is expressed.
there are also global means, through nuclease digest and HPLC separation,
we are now trialling a kit that looks at global DNA methylation levels by Epigentek, it's the methylamp kit.
I think it all boils down that what type of methylation are you looking for. Are you looking at specific gene promoters? then you would look into bisulfite mediated assays. Are you looking genome-wide? The microarrays and MeDIP would be the way to go.
I just attended a great seminar by Steven Jacobsen who studys methylation in plants. I suggest that you look up some of his papers as this will give you ideas about methods, etc. as well as giving you a good background about the study of methylation in plants.
Steven E Jacobsen is one of the biggest gurus in plants epigenetics. his seminars are great. the Jacobsen lab has even got a group in facebook. you can also download his papers in the lab homepage (just google it).
Thanks to all the great suggestions. Now I have a better idea as to where to start.