Do you believe CpG methylation pattern? - (Sep/19/2005 )
I found the promoter sequence of the gene that i am studying from genome brower, and use methprimer design find the CpG island and primers. MSP turned out great results, beautiful bands.
But the problem is: I found all my normal samples so far are partial methlated or totally methlated. But from the published papers, this gene supposed to be hypermethlation in cancer tissue, that means normal should be unmethylated. My boss said methprimer picked up a CpG sites which is belong to CpG island but not involved in gene expression and within the methylation pattern, so this is not a good site to study. I doubet what he said.
First, can we question the published papers?
Second, How can we say that site is not involved in the gene treanscritpion?
Third, Do you think the CpG island has certain Cs before Gs are not related to methylaton study?
MSP only looks at a handful of CpG sites within one assay, without performing bisulfite seqeuncing whereby you can look at almost every CpG within the CpG island, you can not really say that the whole island is methylation by MSP.
The sites that methprimer had picked could well be not involved in gene transcription as your PI said, this is true in some cases where known Sp1 binding sites within the island require to be unmethylated to permit transcription.
There could be other as yet unidentified transcription factor binding sites that are not assayed by a MSP primer set that are essential for gene transcription.
I have also found that the conditions published in a paper may not necessarily work in your own lab and require some tweaking. Maybe if you tweak your MSP conditions (lower the number of PCR cycles) you will see what you are after.
good luck!!
Nick
>>I found the promoter sequence of the gene that i am studying from genome brower, and use methprimer design find the CpG island and primers.
Is the amplicon on the CpG island?
>>But the problem is: I found all my normal samples so far are partial methlated or totally methlated.
Confirm it by bisulfite sequencing which means you need to design another set of primers (BSP).
The sites that methprimer had picked could well be not involved in gene transcription as your PI said, this is true in some cases where known Sp1 binding sites within the island require to be unmethylated to permit transcription.
There could be other as yet unidentified transcription factor binding sites that are not assayed by a MSP primer set that are essential for gene transcription.
I have also found that the conditions published in a paper may not necessarily work in your own lab and require some tweaking. Maybe if you tweak your MSP conditions (lower the number of PCR cycles) you will see what you are after.
good luck!!
Nick
Thanks for the reply, Nick!
You know, the published papers are all based on the sequece from Gene Bank, and When I blast the sequence in Genome.ucsc.edu, I found out that sequence is belong to First intron. And there is a big CpG island within that intron region. But I only found a small CpG island at the promoter/5 upstream by 1000bp, and I designed the primers based on that CpG island. So, Nick, do you think it is right that those old papers study methylation pattern within that intron region? Do you think transcription factor would relate to that region? My PI said nobody has clearify the real promoter region, so that intron part could also be involved in gene expression.
Is the amplicon on the CpG island?
>>But the problem is: I found all my normal samples so far are partial methlated or totally methlated.
Confirm it by bisulfite sequencing which means you need to design another set of primers (BSP).
Hi,PCRMAN
Yes, the amplicon is within the CpG island.
indeed zzylx!
the intronic CpG island could very well control the transcription of the gene and your PI is correct in saying no one clearly knows how CpG islands regulate genes.
Nick
I know for p16 gene, many people have looked into the methylation of CpG islands in the first exon and intron.
dont believe everthing you hear and only half of whata you see
http://www.molecular-cancer.com/content/3/1/22