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type of detection, type of bisulfite conversion, =>different results? - variability between preps and such (Feb/16/2007 )

Hello all,
I am a postdoc on a new topic for me, and have been looking into methylation for about 7-8 months. I have decided that the methyleasy kit, nested PCR, and pyrosequencing are the fastest protocols for me to use to monitor methylation status. But, I have encountered a problem. I am not seeing the variability between samples that I expect. This could be due to a number of reasons, but I would like to know if other people have seen a drastic difference in detected methylation status due to the type of conversion and detection. I can say that my last experiment I treated 8 different samples using the methyleasy kit, I looked at the methylation status of 15 different regions for each sample using pyrosequencing. I am NOT seeing the difference in methylation that is expected, nor am I seeing the same percentage methylation that has been reported for these regions previously. But, I am doing a different protocol. Previously, people in the lab used the modified Former et al bisulfite conversion, pcr, cloned and sequence like 50 clones. Much more labor intensive protocol than what I use. But, different results? Any comments, papers, experiences would be greatly apriciated!!!!

-bunja-

hii bunja,

have you ruled out PCR contamination between your samples?

Also, could it be histone modifications that are changing and not DNA methylation?

-methylnick-

Hi bunja,

I would suggest to perform a mixture experiment with mixes of unmethylated and fully methylated DNA (e.g. Sss1 treatment). Are you using the same primers as your coleagues did for the previous experiments? Did you have complete conversion of non CpG C's? Did you see any differences betwwen different CpG's in your samples or where all exactly the same?

-krümelmonster-

QUOTE (methylnick @ Feb 16 2007, 08:03 PM)
hii bunja,

have you ruled out PCR contamination between your samples?

Also, could it be histone modifications that are changing and not DNA methylation?




Thanks,
I am fairly certain that I am not having PCR contamination between samples. There is variability between samples, just not what is expected. As for the histone modifications, havn't looked into at all. Previous people in the lab found these regions and detected changes in the methylation status due to different treatment groups. When I came in they asked me to streamline the protocol and be able to monitor all these regions through different tissues, treatment groups, ect. So I am in the process of changing it all around. In the end, I know that I am going to have to run a bunch of quality control experiments to test a bunch of parameters to optimize for the correct methylation status.

-bunja-

QUOTE (krümelmonster @ Feb 17 2007, 05:26 AM)
Hi bunja,

I would suggest to perform a mixture experiment with mixes of unmethylated and fully methylated DNA (e.g. Sss1 treatment). Are you using the same primers as your coleagues did for the previous experiments? Did you have complete conversion of non CpG C's? Did you see any differences betwwen different CpG's in your samples or where all exactly the same?



Monster,
I will look into the Sss1 treatment.
I am using some of the same primers, but because of all the different treatment groups and tissues we need to look at, I have developed a way to get clean PCR product for each region using one set of thermocycling conditions. In the end it is fairly slick, I can monitor get 15 different amplicons for pyrosequencing using one set of thermocycling conditions and as low as 0.1ul of bisulfite treated DNA. Now I want to make sure that I am getting the "real" methylation status.

I am seeing compleate conversion of non CpG C's, and there is some variability, just not what we were expecting.

-bunja-

Another possibility may be that PCR my bias towards unmethylated template - often this can be coped with by changing the annealing temp. or the primer design... maybe you don't see differences in your samples due to amplifying only a part of the truth? Hum, just a crude guess wink.gif

Krümel

PS: Mixture experiments should reveal this, too...

-krümelmonster-

QUOTE (krümelmonster @ Feb 19 2007, 04:39 PM)
Another possibility may be that PCR my bias towards unmethylated template - often this can be coped with by changing the annealing temp. or the primer design... maybe you don't see differences in your samples due to amplifying only a part of the truth? Hum, just a crude guess wink.gif

Krümel

PS: Mixture experiments should reveal this, too...



That is exactly what I am looking into now.
1st, I am looking into amount of template to see if this will creat a bias towards unmethylated DNA. I amplified 2 different regions using ~3,2,1,0.5,0.1 ul of bisulfite treated DNA at 20ng/ul. I did this with and without single stranded DNA binding protein (SSB). 2 rationals. First, if the unmethylated DNA is easier to amplify it should be very abundant in the 3,2,1 ul pcr reactions and not so abundant in the 0.5 and 0.1 ul pcr reactions. I would expect that the % methylation in the upper samples to report lower % while the less abundant pcr samples report a higher % methylation. Second, I am testing to see if the SSB will eliminate secondary structure of the bisulfite treated DNA, since there is a theory that the higher the methylation the more secondary structure in the DNA. If this is the case I would expect that the highest % methylation detected for these regions to be in the 1,0.5, and 0.1 ul pcr reactions that contained SSB (the higher DNA concentrations should overwhelm the SSB making unbound DNA available).

2nd, I am running the pcrs at higher temps to see if that helps. I should know in a few days, after I beat people away from the thermocylcler, nested PCR takes a while.

I suppose the next thing I could do is a timecourse with Sss1 and sort of repeat these experiments to see if I can get a higher % methylation. Is that what you mean by a mixture experiment?

Thanks for your imput guys, if you have anymore ideas about perameters I am open for suggestions.

-bunja-

Hi, your plan sounds rationale. There is one paper concerning PCR bias which shows that this can be controlled by including one CpG into the 5' region of the primers ... maybe worth a thought, too.

Fingers crossed,

Krümel

-krümelmonster-

Do you have methylated and unmethylated controls? If you don't want to prepare these yourself you can purchase 5aza2deoxycytidine treated human jurkat DNA (unmethylated) and methylated human jurkat DNA from NEB to ensure your bisulfite conversion is complete.

-MDR-

Do you have methylated and unmethylated controls? If you don't want to prepare these yourself you can purchase 5aza2deoxycytidine treated human jurkat DNA (unmethylated) and methylated human jurkat DNA from NEB to ensure your bisulfite conversion is complete.

-MDR-