MSP result - (Oct/18/2005 )
For the control sample I see a band of "U" primer set, and very very faint (barely see) band of "M" set. For the treated sample I see both "U" and "M" bands clearly. Can I say my gene was hypermethylated after treatment?
how many cycles are your performing in your PCR?
you may have reached teh plateau stage of the PCR and the reaction has been saturated. If you reduce the number of cycles the faint M band would not be present in your control and if your sample becomes methylated afer treatment, your U band in your treated sample will also not be present.
As it stands, because you have both U and M bands in your treated sample you can not categorically say that it becomes methylated, the PCR just needs to be tweaked a little (reducing the number of cycles) and you should get the results that suggest so.
I am so confused about this "black" "white" issue of DNA methylation. Isn't the gene I am looking at could be partially methylated? So I will see band from both "U" and "M".
What do you think about Pyrosequencing? Looks like I will get exact percentage of methylated C in my sample. I am talking to Biotage about this.
certainly you can say that your gene could be partially methylated, however you can't say it's hypermethylated as this usually describes complete methylation in most cases.
In most cases, with CpG islands associated with genes at least, methylation is black and white you either get hypermethylation (full) or hypomethylation (non). This is exemplified in a number of genes involved in cancer progression and the CIMP phenotype described by Issa.
pyrosequencing is a promising technique in that you can measure methylation at a particular site for the whole population of cells in your original sample. It requires the purchase of a machine to do this. I have not had any experience with it as we don't have one on site but it looks promising from what I have read in the literature.