RT PCR for methylation analysis - (Feb/10/2008 )

Hi everyone,

Do you have any personal experience in using RT-PCR for methylation analysis? I mean does this method really work and does it give reliable results? I'm not sure whether it's worth money it will consume..

thanks,
anna

anna there are many different flavours to using RT-PCR for methylation analysis. It is certainly cheaper than bisulfite PCR and sequening.

However you have to think about what you want to find out and what can yield more bang for your buck.

RT PCR methods can evolve around restriction digested DNA using methylation sensitive enzymes which test one maybe more sites within a test amplicon. So you are measuring the amount of digestion from MS enzymes by q-RTPCR.

There is the bisulfite mediated MS-PCR where you are testing the methylation of 3-5 sites within the primer on bisulfite converted DNA, so you have a methylated set and an unmethylated set and run both reactions side-by-side.

There are good review articles on the methods that could be used for methylation and in fact RT-PCR based methods are significantly cheaper than the gold standard bisulfite PCR and sequencing (if you can scale down to 384-format or diluting your master mix)

Nick

thanx a lot, you're really fast


What im' trying to establish is methylation status of a promoter of a certain gene in tumor vs control tissue. so quite a standard work. As for now I would like to state if there are ANY differences - to know if i should spend some more time on this topic or rather give it up and find a new one ASAP.

I'm wondering if you've ever heard of a method based on DMSO, described by N. Kholod ("A New Dimethyl Sulfoxide-Based Method for Gene Promoter Methylation") J Mol Diag. I don't have access to this journal so I haven't read the article yet but the idea sounds interesting.

As for RT, I'm thinking of sth like MethylQuant. I've already done DNA conversion, pcr and sequencing and i'm not satisified with the results. Of course i had to clone pcr products to pGEM - direct sequencing didn't work. I really hate this additional bunch of work and time it takes. RT would be much faster i suppose.

thx
a

The DMSO method I have read and had a go with some standards and was surprised to see it works fine!

I have the paper here and will pm you.

RT will not give you the same resolution as bisulfite sequening would so if you don't pick up anything by RT doesn;t mean the difference is not there, it's just your assay didn't pick it up.

RT is good for screening, so if you know of a site that is differently methylated you can design an RT assay for it and screen lots of samples.



Nick