tough problems on methylation study - (Jun/28/2010 )

hello everyone,

I am now do methylation study on marek's disease. And there are some problems perplex me all the time. Hopefully you can give me some suggestions.

My genes for methylation study came from a piece of gene expression profile. We now plan to study our normal and tumorous tissue. Some researcher suggest me to study the tumorous tissue and adjacent normal tissue. but in my case it's hard to separate normal tissue from tumorous tissue. So will that be a matter if I follow my own thoughts.

how many samples should I conduct at least. This is vary from several to more than 100 according to other references. I planed to do 4 normal and 4 tumorous respectively. Some people suggested at least more than 20 respectively. I am a little confused. Becasue with certian money, to increase samples will decrease our genes for study, then the chance for us to detect the right genes will be less. How about we do less normal and more tumorous samples since in normal tissues the methylation status will not change as significantly as in tumorous samples.

Secondly, which stand of DNA should we choose for methylation study, or should I choose both of the stands because different stands may have different methylated information. Limited references has reported on this.

Finally, how to find the TSS region of a certain gene on Chicken. There are some online program to predict the promoter and TSS regions on human but not for chicken input. I used to think in the genome sequence TSS is at the place where mRNA starts. But my teacher thinks it should locate at the place that precursor mRNA stants, which cannot be found directly on NCBI. So I feel a little lost. Most of the papars mentioned promoter along with TSS and ATG groups. Can I still move on without the annotation of TSS location by only analysing regions at the upstream of ATG groups which is rich of CpGI(S).

Thanks a lot!

Lacee

>>how many samples should I conduct at least. This is vary from several to more than 100 according to other references. I planed to do 4 normal and 4 tumorous respectively. Some people suggested at least more than 20 respectively. I am a little confused. Becasue with certian money, to increase samples will decrease our genes for study, then the chance for us to detect the right genes will be less. How about we do less normal and more tumorous samples since in normal tissues the methylation status will not change as significantly as in tumorous samples.

There is no consensus on how many samples you should analyze. The critical part is that you do not have mutual contamination between normal and tumor. Sample size depends on the observed methylation differences between normals and tumors and variations within each type of samples.


>>Secondly, which stand of DNA should we choose for methylation study, or should I choose both of the stands because different stands may have different methylated information. Limited references has reported on this.

Since methylation is symmetric, usually primers are designed on the sense DNA strand.

>>Finally, how to find the TSS region of a certain gene on Chicken. There are some online program to predict the promoter and TSS regions on human but not for chicken input. I used to think in the genome sequence TSS is at the place where mRNA starts. But my teacher thinks it should locate at the place that precursor mRNA stants, which cannot be found directly on NCBI. So I feel a little lost. Most of the papars mentioned promoter along with TSS and ATG groups. Can I still move on without the annotation of TSS location by only analysing regions at the upstream of ATG groups which is rich of CpGI(S).

I answered this question in your another post.

>>how many samples should I conduct at least. This is vary from several to more than 100 according to other references. I planed to do 4 normal and 4 tumorous respectively. Some people suggested at least more than 20 respectively. I am a little confused. Becasue with certian money, to increase samples will decrease our genes for study, then the chance for us to detect the right genes will be less. How about we do less normal and more tumorous samples since in normal tissues the methylation status will not change as significantly as in tumorous samples.

There is no consensus on how many samples you should analyze. The critical part is that you do not have mutual contamination between normal and tumor. Sample size depends on the observed methylation differences between normals and tumors and variations within each type of samples.

Q: I think you mentioned an important question that no mutual contamination between normal and tumor. Some paper used tumor tissues and adjacent normal tissues. And others used tumor and normal tissues. In my opinion, the first method would be much better if it is easy to separate normal and tumor in a sample. But it's hard for me to do this in my case. On the other hand I think some further exp. need to be conducted to indentify the normal tissue cause tumor tranformation is a time problem. The normal tissue we see through naked eyes may already in the process of transformation. So I finally decide to compare the same tissue from normal individual and infected individual. Is this ok? However, there may be more factors influence gene expression compare to the first method.


>>Secondly, which stand of DNA should we choose for methylation study, or should I choose both of the stands because different stands may have different methylated information. Limited references has reported on this.

Since methylation is symmetric, usually primers are designed on the sense DNA strand.

Q: Sometimes there is hemimethylation. Then the two stands have different methylation information. It is not so popular in human but I am not sure the case in chicken. So I am wondering if it is better for me to analyze the template stand because it is the one that produces mRNA. But if the hypothesis that methylation of DNA affects the transcription factors to combine the TSS because of stereohindrance effect is right, then we should analyze both stands. >>Finally, how to find the TSS region of a certain gene on Chicken. There are some online program to predict the promoter and TSS regions on human but not for chicken input. I used to think in the genome sequence TSS is at the place where mRNA starts. But my teacher thinks it should locate at the place that precursor mRNA stants, which cannot be found directly on NCBI. So I feel a little lost. Most of the papars mentioned promoter along with TSS and ATG groups. Can I still move on without the annotation of TSS location by only analysing regions at the upstream of ATG groups which is rich of CpGI(S).

The resources available for determining TSS is certainly much less for chicken genes than human and mouse genes. You can just find the start of the first exon (or transcription start), the promoter sequence is usually immediately upstream of the TSS. If there is CpG islands in the vicinity, it is pretty much sure that is the promoter sequence

Q: So it is ok for me to just find ATG Groups or CAT Groups first, then go upstream of these region and look for CpGI(s).

Q: I think you mentioned an important question that no mutual contamination between normal and tumor. Some paper used tumor tissues and adjacent normal tissues. And others used tumor and normal tissues. In my opinion, the first method would be much better if it is easy to separate normal and tumor in a sample. But it's hard for me to do this in my case. On the other hand I think some further exp. need to be conducted to indentify the normal tissue cause tumor tranformation is a time problem. The normal tissue we see through naked eyes may already in the process of transformation. So I finally decide to compare the same tissue from normal individual and infected individual. Is this ok? However, there may be more factors influence gene expression compare to the first method.

I agree with you that adjacent tissue may not be normal at all. If you just compare tumor tissues from patients with normal tissues from normal individual, no one would say you are wrong.

Q: Sometimes there is hemimethylation. Then the two stands have different methylation information. It is not so popular in human but I am not sure the case in chicken. So I am wondering if it is better for me to analyze the template stand because it is the one that produces mRNA. But if the hypothesis that methylation of DNA affects the transcription factors to combine the TSS because of stereohindrance effect is right, then we should analyze both stands.

Yes, there is sometimes hemimethylation. If you really want to look into that, that's perfectly fine. To do that, you need to use the minus strand to design a different set of primers.

Q: So it is ok for me to just find ATG Groups or CAT Groups first, then go upstream of these region and look for CpGI(s).

No, ATG does not always stay close to transcription start site. If there is no other way of predicting transcription start site, looking into the region upstream ATG should be fine. But at least you can use the upstream sequence to predict if there are CpG islands.

Ok thanks so much for your help. All the best!

pcrman on Jul 2 2010, 08:19 AM said: