Is it absolutely necessary to do BGS before doing MSP for new genes? - (Nov/02/2006 )
Hi there,
Just want to get some advice for something I am hoping to do.
Is it absolutely necessary to do BGS in order to use MSP to correlate the down-regulation of genes?
These genes that I will be working on are relatively new and nobody have ever worked on the possibility of methylation silencing on them before. I only have the databases to pull out useful information like the promoters and exons location, CpG islands, etc. I intend to use the fastest time possible to correlate the down-regulation of these new genes using MSP, but hope to skip doing BGS as it is really time consuming. Can anyone give me some advice? Many thanks in advanced!
Darth 
hi Darth,
In my experience, it would be better to perform bisulfite genomic sequencing (BGS) prior to performing MSP. This is because MSP only assays CpG's the primers test which range between 3-5 within the entire promoter and then from this the methylation status of the entire promoter is extrapolated.
In your case, because it is an unknown gene, promoter, regulation of gene, it would be more appropriate to perform BGS albeit more time consuming, at least you would test every CpG site and you may pick up the one or two critical CpG's that were responsible for the regulation of the gene, something that could be missed in a typical MSP assay.
good luck with it!
N
Hi Nick,
Thanks for your fast response. I get what you mean. I was in a hurry as I thought of adding these genes into a panel of genes that are methylated in prostate cancer for cancer detection by quantitative real-time MSP. If I have to do BGS, how many cell lines should I do in order to have a good gauge of where the "important" CpG sites are? Do you think 3 is enough? 2 cancer cell line with one normal DNA control. And how many colonies for each at least?
Sorry for the many questions I have as I'm quite new to this field. And thanks again for your reply.
Darth
In my experience, it would be better to perform bisulfite genomic sequencing (BGS) prior to performing MSP. This is because MSP only assays CpG's the primers test which range between 3-5 within the entire promoter and then from this the methylation status of the entire promoter is extrapolated.
In your case, because it is an unknown gene, promoter, regulation of gene, it would be more appropriate to perform BGS albeit more time consuming, at least you would test every CpG site and you may pick up the one or two critical CpG's that were responsible for the regulation of the gene, something that could be missed in a typical MSP assay.
good luck with it!
N
a normal and a cancer line should be enough but it doesn't hurt to do more.
the more you sequence the better the outcome, minimal, I have seen in papers 8-12 clones, but i am sure more were sequenced and were not published.
good luck!
Nick
Hi Nick,
Thanks for your reply. What you suggested make sense and I know what to do now. Will try doing what you suggested.
Darth
If I have to do BGS, how many cell lines should I do in order to have a good gauge of where the "important" CpG sites are? Do you think 3 is enough? 2 cancer cell line with one normal DNA control.
a normal and a cancer line should be enough but it doesn't hurt to do more.
the more you sequence the better the outcome, minimal, I have seen in papers 8-12 clones, but i am sure more were sequenced and were not published.
good luck!
Nick