CpG site or CpG island, which is important - (Jun/02/2005 )
>>When I ask control, I mean the internal gene contol (Say, actin) to quantify the relative gene expression level of my target gene in QPCR. But I wonder global methylation modifier may change the expression level of actin too, which will make the relative expression level unsound.
It is possible that actin expression is affected by certain treatment. There are some papers describing such phenomenon, such as this paper beta-Actin—an unsuitable internal control for RT-PCR .
Alternatively you can use 18S rRNA as internal control. Ambion site has a nice page explaining this http://www.ambion.com/techlib/tn/83/839.html
-pcrman-
QUOTE (labtechie @ Jun 8 2005, 08:44 AM)
Basically, I thought I had proven what you said, pcrman, about most CpGs outside of CpG islands being methylated and not showing any kind of change in cancer. However, this promoter does show only a 40-50% methylation (Bisulfite sequencing, 10 clones, from normal human genomic DNA) in certain CpGs, then 80-100% methylation in the cancer (Bisulfite sequencing, 25 clones, cell lines, unfortunately, but it wasn't my choice...I'm just a tech). It's statistically significant, but I don't believe it's earth-shattering. I think my results are just going to be a sentence or two in the paper about characterization of the promoter we are studying. Unlesss you think it could mean something real? Nick I know you are proponent of this theory....
Hi labtechie, I think you have done a great job helping characterize the promoter. I have some questions. How did you get the percentage numbers 45-50% and 80-100%. Are these number represent a particular methylated CpG site or any methylated CpG site in the amplified sequence? What criteria did you use for CpG island prediction and how many CpG sites are in your amplified region.
-pcrman-
QUOTE
Hi labtechie, I think you have done a great job helping characterize the promoter. I have some questions. How did you get the percentage numbers 45-50% and 80-100%. Are these number represent a particular methylated CpG site or any methylated CpG site in the amplified sequence? What criteria did you use for CpG island prediction and how many CpG sites are in your amplified region.
Hi pcrman-
Thank you so much for your help! Going back to my original question, these are individual CpG sites in a promoter that does NOT qualify as a CpG island. There were 6 CpGs in my amplicon, and three of them show significant difference in methylation status between normal and cancer, as measured by cloning and sequencing. The percentage numbers are simply percent of clones (n=10 for normal and n=12 and n=13 for the two stages of cancer I studied, which I also combined into a larger group of just cancer n=25) that are methylated at a particular site, taking each site individually.
What do you think?
labtechie
-labtechie-
QUOTE (labtechie @ Jun 9 2005, 06:20 AM)
Going back to my original question, these are individual CpG sites in a promoter that does NOT qualify as a CpG island. There were 6 CpGs in my amplicon, and three of them show significant difference in methylation status between normal and cancer, as measured by cloning and sequencing. The percentage numbers are simply percent of clones (n=10 for normal and n=12 and n=13 for the two stages of cancer I studied, which I also combined into a larger group of just cancer n=25) that are methylated at a particular site, taking each site individually.
What do you think?
labtechie
What do you think?
labtechie
I would say that is very interesting. Are there any CpG island or CpG rich area nearby? Based on published literature, if a gene doesn't have a CpG island in its promoter, it is usually unmethylated in cancer. Two such examples are TP53 and PTEN, both are tumor suppressor gene.
-pcrman-