7 replies to this topic

[epigenetics]

I am facing some problems in MSP.
I have two samples from two patients and one gene to look for. In one, The primers are working perfectly. But in other, not.
What might be the reason? My DNA quality is very good for both of my samples. Bisulfite should work properly too. Controls are working perfectly. i cant find out the reason. Can it be like this , if there is difference between "C" residues which are methylated , suppose in sample 1 , C at 12 is methylated, but in other C at 5 is methylated. so my primers are not working with 2 nd one.
is it possible that in two individuals with same disease, in same CpG island, the methylated residue is different?
Anybody has any experience?

[emacalate]

So in the other sample which doesn't amplify, both meth and unmeth primers dont work? Have you tried increasing your template concentration I found that worked for me when doing MSP in the past. ( Ive went up to 100ng/rxn I think) But yes in the same CpG island for a different sample the methylation could be elsewhere, bisulphite sequencing will allow you to  look at more CpGs.

[epigenetics]

Thanks for the answer.
I used also 100ng/ PCR reax. I can try with more and My samples are ok as i got result for another gene in both of my samples.
We are also planning to move to sequencing, So did you try PCR followed by sequencing without cloning. In this case, we will do sequencing by capillary sequencer. If you have, how do you analyse results, i mean which software you use and how much complex the result will be?
And what are the problems you faced in Cloning followed by sequencing or in Sequencing directly?

emacalate, on Feb 25 2009, 08:44 PM, said:

So in the other sample which doesn't amplify, both meth and unmeth primers dont work? Have you tried increasing your template concentration I found that worked for me when doing MSP in the past. ( Ive went up to 100ng/rxn I think) But yes in the same CpG island for a different sample the methylation could be elsewhere, bisulphite sequencing will allow you to  look at more CpGs.

[Davo]

I've performed direct sequencing of Bisulfite PCR products and found it to be quite reproducible. I analysed the results in ABI Seq Scanner ver 1 (google it and download) and then measured the C and T peak heights at every CpG position. Do the calculation and whalla, you have some results. Very tedious if you have numerous samples/genes, but also very reproducible. Cloning will give you information about individual alleles of the DNA, but you will need to do several reactions for each sample to get an idea of what is going on at that section of the DNA.

Best of luck!
Dave

[epigenetics]

Hey thanks Dave,
Do you use Meth Primer to generate BSP primers? Is it giving good outcome for most of your genes?
And by "CpG positons" did you mean CpG islands? So if your primer is amplifying 2 or more CpG islands , in that case you are measuring C and T peak heights at every CpG island? correct me if i am wrong?

Davo, on Mar 1 2009, 03:26 PM, said:

I've performed direct sequencing of Bisulfite PCR products and found it to be quite reproducible. I analysed the results in ABI Seq Scanner ver 1 (google it and download) and then measured the C and T peak heights at every CpG position. Do the calculation and whalla, you have some results. Very tedious if you have numerous samples/genes, but also very reproducible. Cloning will give you information about individual alleles of the DNA, but you will need to do several reactions for each sample to get an idea of what is going on at that section of the DNA.

Best of luck!
Dave

Edited by epigenetics, 02 March 2009 - 07:23 AM.

[Davo]

I don't use Meth Primer. I get the sequence of the area of interest from NCBI and then design by eye. By 'eye' I mean I put the sequence into MS Word, highlight all the CpG's with red (can't put a primer there) and then convert all non-CpG cytosine to T, and make them bold (Want to include a few of these in the primer). I had some mixed results with this. Some genes are more difficult than others if they are CpG rich, in which cases you can't find a good 20bp to put a primer. My fragments were up to 600-700bp at most, and I could get around 500bp of sequence from them.

In terms of direct sequencing most of it worked well, but some genes would not sequence, despite the PCR working well. Cloning would get you around this problem if your sequencing primer was located on the plasmid rather than the amplicon. I put the problem down the chemistry of the reaction and inappropriate sequence of the primer.

I measured every CpG site within the amplicon, but not the entire CpG island as the island was usually larger than my PCR fragment. The CpG sites around the transcriptional start site are most important in my opinion

[epigenetics]

Thanks a lot.

Davo, on Mar 3 2009, 05:50 PM, said:

I don't use Meth Primer. I get the sequence of the area of interest from NCBI and then design by eye. By 'eye' I mean I put the sequence into MS Word, highlight all the CpG's with red (can't put a primer there) and then convert all non-CpG cytosine to T, and make them bold (Want to include a few of these in the primer). I had some mixed results with this. Some genes are more difficult than others if they are CpG rich, in which cases you can't find a good 20bp to put a primer. My fragments were up to 600-700bp at most, and I could get around 500bp of sequence from them.

In terms of direct sequencing most of it worked well, but some genes would not sequence, despite the PCR working well. Cloning would get you around this problem if your sequencing primer was located on the plasmid rather than the amplicon. I put the problem down the chemistry of the reaction and inappropriate sequence of the primer.

I measured every CpG site within the amplicon, but not the entire CpG island as the island was usually larger than my PCR fragment. The CpG sites around the transcriptional start site are most important in my opinion

[et2b]

epigenetics, on Mar 4 2009, 10:12 PM, said:

Thanks a lot.

Davo, on Mar 3 2009, 05:50 PM, said:

I don't use Meth Primer. I get the sequence of the area of interest from NCBI and then design by eye. By 'eye' I mean I put the sequence into MS Word, highlight all the CpG's with red (can't put a primer there) and then convert all non-CpG cytosine to T, and make them bold (Want to include a few of these in the primer). I had some mixed results with this. Some genes are more difficult than others if they are CpG rich, in which cases you can't find a good 20bp to put a primer. My fragments were up to 600-700bp at most, and I could get around 500bp of sequence from them.

In terms of direct sequencing most of it worked well, but some genes would not sequence, despite the PCR working well. Cloning would get you around this problem if your sequencing primer was located on the plasmid rather than the amplicon. I put the problem down the chemistry of the reaction and inappropriate sequence of the primer.

I measured every CpG site within the amplicon, but not the entire CpG island as the island was usually larger than my PCR fragment. The CpG sites around the transcriptional start site are most important in my opinion

MSP primers are completely reliant on binding efficiency to give reliable results, maybe the patient has a SNP? But I would drop MSP for ABI. Just as easy and gives and more reliable data than MSP. MSP is quite shitty, no really.