Positive bands in both M and U for MSP - have tried everything - please help! (Jul/24/2007 )
I am carrying out MSP on a set of cell lines and clinical samples. My primers have been previously published for methylation, along with the PCR conditions. I tried these on some modified genomic DNA from cell lines and it worked perfectly, also the methylation status correlated with expression levels of my protein.
I decided to go ahead and modify all my clinical samples and go ahead with MSP on these, but I ran the gel and got really bright bands with both the methylated and unmethylated primers, in everything, even my positive and negative controls!
I have repeated this many times with the same results. I have used new enzyme, buffer and MgCl2, made up new primers, dNTPs, everything I can think of. I remodified some of the samples in case the modification went wrong, but got the same results. I gave the DNA I had modified to a lap colleague who ran them with his primers, and they worked, so I dont think it is my modification. I also changed the MgCl2 concentration, to no avail, although it had worked before at the original concentration.
I cant figure out what is going wrong, I have spent weeks repeating the same PCR and I am going a little crazy, I also have run out of ideas of what parameters I can change to fix this. Please help! I hate science
Can you post the primer sequences and your cycle conditions? That'll help make the picture clearer.
I take it you are using tumor samples, could these samples contain a mixture of normal and cancer cells for which you have amplicons with both primer sets?
Hi, thanks for the help. The primer sequences are:
and the conditions are:
1 - hotstart: 94degrees for 12 minutes
2 - 94degrees for 30sec
3 - 54degrees(unmeth)/57degrees(meth) for 30 sec
4 - 72degrees for 30sec
5 - repeat steps 2-4 35 times
6 - 72degrees for 7 minutes
And although the samples I am using are tumour samples, this is also happening in my cell lines and positive and negative controls, which previously were working fine.
Well I don't see obvious flaws in your primer sequences and, as you told us, they worked fine in the beginning. Is contamination possible?
To be honest, this doesn't really surprise me. I get these results, too and I have been able to confirm them by direct sequencing. Should I be worrying about this? I thought I simply had a heterogenously methlyated (tumor) cell population...
Cyburn, I guess you are rigth about that.
Methfairy, you could still try to use real-time MSP to look for metylation differences... or use a quantitative method like BSP or Pyrosequencing.
Thanks for all your help. I have tried a couple of things which I thought might solve the problem if it was contamination, like using new primers, new reagents, and remodifying my control samples. However, when I do MSP on these samples I get exactly the same result, positive bands with both methylated and unmethylated primters! I have noticed that the band I get in my unmethylated samples is a little weaker than the methylated, not sure if this means anything. I have run out of ideas, so am thinking of going down the sequencing route even though this means a lot of work, sequencing 8 clones from each sample.., and I have about 60 samples... If anyone has any other ideas as to what could be going wrong, please post them. Thanks a lot for your help!
Methfairy losing faith in MSP
It depends on your question. If it is okay, try to establish real-time MSP (i.e. use a real-time cycler and SYBR green). You can calculate %methylation from the ct-values. It should be okay if you calibrate your assay with mixes of methylated and unmethylated DNA.
I guess - again, this depends on your approach - you could also go for direct sequencing. You would see both a C and a T peak at the appropriate locations. That's my approach, as I'm only interested in a very rough characterization (methylated, unmethylated, heterogeneously methylated). If you like, I could upload a screenshot of such a heterogeneously methylated CpG from direct sequencing.