MSP and Bisulfite genomic sequencing results don't match! - (Jun/27/2006 )

Hi

I have found that my MSP and BGS results don't match up! For example, for a gene x of interest in HeLa cells, using MSP I see a predominant methylated band and a weak unmethylated band. I have now also perfomed bisulfite genomic sequencing of the whole promoter region of this gene down to the second exon in HeLa cells, covered by six primer sets. However the CpGs that my MSP primers covers are all unmethylated by sequencing? Can anyone help explain this? am I missing something really obvious? I would appreciate any advice on this!!!! Many thanks.

what are your cycling conditions for MSP?

Also what primers are your using for BSP, are they distinct from your MSP set?

How many clones have you sequenced for BSP to determine that you have such a descrepancy.

[quote name='methstudent' date='Jun 27 2006, 05:03 AM' post='57299']
Hi

I have found that my MSP and BGS results don't match up! For example, for a gene x of interest in HeLa cells, using MSP I see a predominant methylated band and a weak unmethylated band. I have now also perfomed bisulfite genomic sequencing of the whole promoter region of this gene down to the second exon in HeLa cells, covered by six primer sets. However the CpGs that my MSP primers covers are all unmethylated by sequencing? Can anyone help explain this? am I missing something really obvious? I would appreciate any advice on this!!!! Many thanks.
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I had the same experience with you two months ago!I think that MSP easily creats false positive, for i usually have the M-bands with the healthy people'MNCs.I suggest that you repeat your MSP.

linfuan006

Dear MethylNick

as Linfuan said, it could be false positives because the M bands are more likely to amplify compared with the U bands, by their very nature in base composition, there maybe inherent biases to which template is amplified in MSP. M bands have CG in them and thus the Tm of the product is higher than the same product that is unmethylated. Another issue is the number of cycles you are using, becuase the M band is favoured over the U band it seems in your case, 40 cycles is saturating the PCR and you get the M band coming up strongly, if you try and reduce the nnumber of cycles to say 20-25 you may find that the U band predominates cf. the M band at that stage of the PCR.

10 clones is the minimum that should be sequenced in an ideal situation and the fact that you see only unmethylated templates suggests your MSP is not optimum.

Nick