Weak amplification in Methyl specific PCR (MSP)

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Firoz Ahmad
Firoz Ahmad's picture
Weak amplification in Methyl specific PCR (MSP)

Hello everyone
I am using your CpG WIZ MGMT amplification kit for gliomas cases. Currently I am standardizing the assay and I have followed the instructions mentioned in the kit insert. I am using Qiagen DNA kit for extraction of DNA from FFPE tissue . I used 15 ul (approx 1 ug) of the FFPE DNA for bisulfite conversion using Epitect kit from Qiagen and did the PCR amplification . The gel pic post PCR amplification shows that except for the control DNA (universal methylated DNA) the samples did not showed any amplification with very very fain amplification with unmethylated primers while with methylated primers no amplifcation was observed. Can you please suggest me how to improve the amplification intensity of the amplicons?

The input DNA samples looked of without much degradation and I feel that it is unlikely that the DNA is degraded upto that extent that it cannot under go chemical modification. Even the modification was done one day prior to PCR.

Looking forward to hear from you at the earliest.

Thank you all in advance for the suggestions

Ivan Delgado
Ivan Delgado's picture
Hi Firoz,

Hi Firoz,

I have some experience PCR amplifying methylated DNA (well, DNA that was bisulfite converted before PCR). Unfortunately I cannot give you a magic bullet about this because of the following two reasons: 

1. Bisulfite conversion is a tricky experiment that is not easy to master. The fact that you are getting the control to work well suggests your DNA may not be working as well. If you are not already doing so you may want to get better quality DNA (purified using a Qiagen kit). 

2. DNA extracted from FFPE is known to be of bad quality (many times already degraded). In other words, you are trying to perform a hard experiment using one of the worst sources of DNA. My recommendation is to get this protocol to work on DNA that came from another tissue source (fresh tissues), to make sure the problem does not come from the FFPE itself.

Good luck

Firoz Ahmad
Firoz Ahmad's picture
Thanks Ivan for your reply. I

Thanks Ivan for your reply. I would like to share with you about todays troubleshoot. I doubled the quantity of input DNA before PCR and added 3mM MgCl2 instead of 1.5 mM. I observed a significant improve ment in the intensity of the band. This was very encouraging. Would you like to add any of yr comments for further improvement?
Thanks
Cheers

Ivan Delgado
Ivan Delgado's picture
Hi Firoz,

Hi Firoz,

That indeed sounds encouraging. Here is another suggestion: 

While this takes more work, it may actually get you were you need. Set up the same PCR reaction in 2 to 4 tubes and at the end of the reaction mix all tubes together. Then purify the PCR using a PCR reaction purification kit. At the end of the purification resuspend/elute the PCR using one half to one fourth of the total volume of your PCR reactions. You should get more than enough PCR amplicon this way. 

Firoz Ahmad
Firoz Ahmad's picture
Thats a nice suggestion.

Thats a nice suggestion. However I was wondering wil this be a acceptable way to pull down 2-4 tubes into a single tube, will this strategy wont add any bias for reporting the case to be a positive case?
The assay is a qualitative test for clinical samples

Ivan Delgado
Ivan Delgado's picture
In that case, I am not sure

In that case, I am not sure what to suggest. I do not think that one can reliably test, for clinical purposes,  bisulfite treated FFPE samples. It is just too much to ask. 

Having said that, if you can first show that your assay works very well on bisulfite treated DNA from fresh tissues, and then show that the DNA from your FFPE sample is good enough for PCR amplification after bisulfite treatment using a PCR assay that is known to work well, then maybe you can try to optimize your assay for this purpose. All I am saying here is: you need controls for every step of your protocol.

Firoz Ahmad
Firoz Ahmad's picture
Thanks Ivan, Well this assay

Thanks Ivan, Well this assay is being offered bye all international labs for deciding therapy in brain tumors. All the lab s use FFPE tissues. As far as the controls are concerned, I take appropriate positive controls however the issue is less intensity bands.

Ivan Delgado
Ivan Delgado's picture
Hi Firoz,

Hi Firoz,

Anytime. If the assay is already a well established one used by a large number of people, then the issue is with your DNA. As I said earlier it is not uncommon to have FFPE DNA degrade. My recommendation, as I also said earlier, is to test the assay using other sources of tissue to determine if indeed the problem you are having is with your FFPE derived DNA. Just extract, and bisulfite treat, DNA from fresh tissues and see if you get better amplification. 
Other than that I cannot think of another suggestion. 

Good luck