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Inconsistent MSP results


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#1 FFPE1

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Posted 15 January 2010 - 06:51 AM

Hi,

I am having problems with MSPs that I hope someone can help me with please.... I cut formalin fixed paraffin embedded sections and then did laser capture microdissection on these sections to isolate specific cell types in 5 control patients and 5 diseased patients. Epithelial cells and lamina propria were extracted separately for each patient and gDNA was extracted from the laser capture microdissected samples.

After gDNA extraction I calculated DNA conc and the purity was also ok. I did a regular PCR with a housekeeping gene and I only proceeded with the samples in which I amplified a specific product for the houosekeeping gene (~ 500bp). I then bisulfite converted this gDNA (approx 150 ng) so that I could do MSP on these samples. P16INK4a (Herman et al., 1996) and Mint1 (Lee et al., 2004) were two of the genes that I examined. The MSP primers were those from the papers I referenced and I double checked these primers and cycling conditions for MSP using universally methylated and universally unmethylated DNA. The MSPs for these two genes were working very well on the universally methylated and unmethylated DNA with the respective primers.

However, when I did MSP on the gDNA that I extracted after laser capture microdissection I got some very strange results... For P16 on the 5 control patient samples I got very weak if any bands in the MSP for both methylated and unmethylated alleles... For P16 on the 5 diseased patient samples I got very strongly methylated alleles and some weak bands for unmethylated. I anticipated this result for the diseased patient samples but I do not understand why I didn't get any bands in the control patient samples.... Could it be due to incomplete bisulfite conversion? I am going to check this by doing a regular PCR with the wild type PCR primers... However, I don't think this is the case as all samples were bisulfite treated at the same time and the p16 MSP on the lamina propria dissected control samples worked fine.... Also, if this was the case would there not be a bias toward amplification of the methylated alleles? And I did not see this in the controls, I just didn't get any amplification in some cases.

I then did MSP using Mint1 MSP primers on the same patient samples (also published primer sequences). The MSP was working well with the positive control samples, however on the patient samples I did not get any bands at all!! The samples were all extracted at the same time and then aliquoted so should not be degraded either due to freeze thaw...

Has anyone any idea what could be happening?? I am relatively new to this area and as the MSP primers are validated I don't think these are the problem.

I spent a long time getting these samples by laser capture microdissection and only have a small amt of each sample so I really appreciate any help/suggestions anyone may have on this. Thanks

#2 Grake

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Posted 15 January 2010 - 07:24 AM

How did you Bisulfite your DNA? Manually or using a kit. In my former lab, we just saw that the manual conversion got all the bad points... labor intensive, need more DNA, less consistent.... So, if you use a manual conversion, just go with a kit. There are not so expensive compare to the benefits... Moreover 150ng is not a lot for your bisulfite, according that the conversion yield is never 100%. I don't how much of the converted DNA you used for your PCR, but maybe it is not enough.

Good luck.

#3 FFPE1

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Posted 15 January 2010 - 07:31 AM

How did you Bisulfite your DNA? Manually or using a kit. In my former lab, we just saw that the manual conversion got all the bad points... labor intensive, need more DNA, less consistent.... So, if you use a manual conversion, just go with a kit. There are not so expensive compare to the benefits... Moreover 150ng is not a lot for your bisulfite, according that the conversion yield is never 100%. I don't how much of the converted DNA you used for your PCR, but maybe it is not enough.

Good luck.



I used the qiagen epitect bisulfite kit to bisulfite convert my DNA. I am new to the lab but it was used by previous members of the lab and worked well. But they did have much higher amt's of stating DNA to work with... For my PCRs I used 4 ul of the bis converted DNA. The thing is it was enough for some samples but not all and I started off with a similar amt of DNA for all patient samples....

#4 MoB

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Posted 16 January 2010 - 05:29 AM

FFPE is a tricky sample. The DNA yield after extraction depends on the method of fixation and esspecially the age of your samples. But I agree to Grake, 150ng is a very small amount. After bisulfite treatment and purification your final concentration of amplifyable DNA should be very low. You could increase the yield after purification by conducting the modiefied Qiagen protocol for FFPE-samples or/and you add a carrier to your sample.

Hope that helps

MoB

#5 FFPE1

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Posted 18 January 2010 - 02:21 AM

FFPE is a tricky sample. The DNA yield after extraction depends on the method of fixation and esspecially the age of your samples. But I agree to Grake, 150ng is a very small amount. After bisulfite treatment and purification your final concentration of amplifyable DNA should be very low. You could increase the yield after purification by conducting the modiefied Qiagen protocol for FFPE-samples or/and you add a carrier to your sample.

Hope that helps

MoB



Hi,

Thanks for your suggestions. I did the modified Qiagen protocol for FFPE samples and added carrier RNA to my samples to try and maximise the yield with only 150 ng of input DNA. So do you think the reason for the inconsistent MSP results are likely due to low concentrations of amplifiable DNA rather than problems with the bisulfite cnversion etc..?

#6 Grake

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Posted 21 January 2010 - 12:01 PM

Since you use a kit for the bisulfite conversion, I think you should not have any problem with it. If you still want to verify your conversion, you can just try to amplify a sequence of DNA before conversion. I mean try to amplify something like you didn't bisulfite your samples. If you got something you can consider that it is not good, but that will mean that you still have completely untreated DNA (the probability is near 0). You can also try to sequence you bisulfite DNA with primer non specific for methylation (without CG in their sequences). If you do so and obtain heterozygous at level of thymin it is not good.
But according to the quantity of template you put before conversion and the fact that you use a kit... I really doubt your problem is the conversion.

I didn't realize that you used FFPE samples!!! Because the other thing is FFPE protocols degrade the DNA (cut it in small piece...). and it is more difficult with this kind of DNA to get amplification product quite long (more than 250bp). It will really depend on each samples. In my former lab, (where we did a lot of PPFE tissue), we performed a multiplex PCR with different amplicons up to 500bp to see the maximum length that you can amplify.

#7 FFPE1

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Posted 22 January 2010 - 01:57 AM

Since you use a kit for the bisulfite conversion, I think you should not have any problem with it. If you still want to verify your conversion, you can just try to amplify a sequence of DNA before conversion. I mean try to amplify something like you didn't bisulfite your samples. If you got something you can consider that it is not good, but that will mean that you still have completely untreated DNA (the probability is near 0). You can also try to sequence you bisulfite DNA with primer non specific for methylation (without CG in their sequences). If you do so and obtain heterozygous at level of thymin it is not good.
But according to the quantity of template you put before conversion and the fact that you use a kit... I really doubt your problem is the conversion.

I didn't realize that you used FFPE samples!!! Because the other thing is FFPE protocols degrade the DNA (cut it in small piece...). and it is more difficult with this kind of DNA to get amplification product quite long (more than 250bp). It will really depend on each samples. In my former lab, (where we did a lot of PPFE tissue), we performed a multiplex PCR with different amplicons up to 500bp to see the maximum length that you can amplify.



Thanks for your ideas. I have ordered wild type primers that correspond to the gene specific MSP primers. This will tell me if the bisulfite conversion is the problem. Although based on some more expts I have done this week I don't think this is the problem.

Yes, using the FFPE samples is difficult. But I amplified a 500bp housekeeping gene before bisulfite conversion to check the quality of my gDNA after fixation and laser capture microdissection. If I amplified the 500 bp housekeeping gene, I proceeded to bisulfite conversion. I know it is only one gene but it gave me an idea of the quality of the DNA without having to waste alot of my microdissected samples. Also, all of my MSP primers amplify up < 150 bp so it should not be as much of a problem here.

I have also looked at the same MSPs using fresh tissue from the same samples and all the MSPs worked really well, so it is most likely the template DNA from FFPE and microdissected samples that is the problem....
Very frustrating!

#8 Grake

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Posted 04 February 2010 - 03:03 PM

Hi FFPE1,

I found something that should interest you, concerning the bisulfite conversion efficiency...
With the ideas I gave you we don't go far enough...
In their paper ("Methylight" in Methods in Molecular Biology 2009 PMID:18987824) the team of Peter Laird describes an assay to really assess the bisulfite efficiency.

You're not oblige to use methylight (which is a method that I dislike), but you should take a look to their quality control #3. They found a sequence in the genome that doesn't change with bisulfite treatment (at the level of primers). this way your amplification is not dependent on your bisulfite treatment and you can check the cytosines in your amplicon to assess your conversion rate.

good luck!




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