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Question: Start with genomic DNA or mRNA for methylation analysis - (Feb/15/2011 )

I am about to start a project looking at the methylation status of some candidate genes. I need to explore the methylation profile of the promoter region, when I read paper, some people use mRNA and some start with genomic DNA, may anybody give me the advice on what should be the starting material for doing so.

What is the difference between using these two. I think I should use genomic DNA as the promoter is untranslated, but someone tell me I should use mRNA instead since, the promoter region is also include within exon. I am lost and need suggestion. Thank you :unsure:

Maran

-Maran-

I am afraid you have confused two things: Whether you should design primers on mRNA coding region or you should use mRNA as the material for methylation analysis. The answer to the first question is that you can study the promoter region as well as the mRNA coding region especially the first few exons and introns because methylation may extend into the coding region. The answer to the 2nd question is you should only use genomic DNA to study DNA methylation. methyl-cytosine only occurs in DNA.

-pcrman-

Thank you so much for your help, and also,may you kindly suggest me how to predict the promoter region? As I have read some previous post on how to predict the promoter region, and you gave very clear instruction, and now I have some more question to ask

1)When I align the CDS with the mRNA or genomic sequence, some time the transcription starting site is located in the 3 exon, in this case, should I assume the promoter is lying within the exon 1 & 2 as well as the -500 5'UTR?

2)If so, should the intron 1 & 2 be included for the prediction of promoter? or only exon be regarded as promoter

3)If form those promoter prediction programme indicate there is no CpG Island, does it mean methylation will happen?

Because I am only a fresh MPhil, so even I read some books and paper, I could not make myself clear

Thank you.

Maran

-Maran-

1)When I align the CDS with the mRNA or genomic sequence, some time the transcription starting site is located in the 3 exon, in this case, should I assume the promoter is lying within the exon 1 & 2 as well as the -500 5'UTR?

>>You have to determine where exactly is the first exon by looking into different databases. Probably the CDS you get does not represent the right mRNA sequence of the gene and that is why it aligns to the 3rd exon. Can you give the name of the gene?

2)If so, should the intron 1 & 2 be included for the prediction of promoter? or only exon be regarded as promoter

>> See above

3)If form those promoter prediction programme indicate there is no CpG Island, does it mean methylation will happen?

>>This is a hard question. originally it is believed that methylation of CpG island affects gene transcription. Now methylation in gene body and non-cpg island region also regulates gene expression.

-pcrman-

Hello pcrman

Thank you very much for your information, for example, the human FHIT gene (NCBI Reference Sequence: NM_002012.2). Its CDS started at 372 in exon 5. In this case, how could I deduce its promoter sequence? Should I take the ~600bp upstream of the exon 1 as the promoter region?

-Maran-