MSP - MSP (Nov/11/2005 )
This is my first time posting so please be gentle
I am looking to set up a MSP experiment to establish whether or not my candidate genes are slienced by methylation in my tumour samples. I have been doing alot of reading and am fairly confident about the principles of MSP. To test myself I have looked at the MSP primers and gene sequences others have used in publications taking into account promoter, exon and predicted CpG island locations.
I have frequently noticed that the MSP primers together span at most 9 CpG sites and the product is a small size compared to that of the promoter region and corresponding CpG island. In some cases these products were sequenced to establish the methylation state of other CpG sites between the MSP primers but not in all cases.
Is this enough information about the methylation status of a promoter region to state that promoter methylation has silenced a gene?
I planned to do MSP followed by (CGCG) BstUI restriction digest. Do you think this would be sufficient to establish the methylation state of a promoter if the whole promoter is looked at by the MSP primers (or multiple sets).
Sorry its long winded, I am just confused over how only MSP over a small region of the promoter has been used to determin the methylation state of a promoter.
Thanks for your time.
I think you are on the right track.
One think I want to point out is that digesting MSP product by BstUI makes no sense, because MSP products either from methylated or unmethylated primer pair are biased PCR products. The M product will have more methylated molecues if not all than the U product.
MSP can only tell you the methylation status of the CpG sites where the primers bind to. If the gene you are interested in has not been studied before, you better first do a bisulfite genomic sequencing analysis to determine methylation of which CpG sites is important for transcription and then you can use MSP to screen large number of samples for methylation at those sites.
I second pcrman,
in addition, for promoters it is generally assumed that MSP primers to promoters will reflect the general methylation status of the whole promoter. however, there will be exceptions as all you need is to inhibitor transcription factor binding within the promoter and all this needs is methylation of the binding site (while the rest of the promoter is unmethylated).
It would be pruedent to look at the entire promoter for methylation status and then screen your samples by MSP.