DNA hypo-methylation analysis - Check if a CpG island is hypomethylated (Sep/01/2004 )
We are studing a gene whose expresssion is up-regulated in cancer cell lines. Shortly upstream of it, there is a 600 bp CpG island and we would like to check if it gets undermethylated in the over-expressing cell lines. We are aware that a Sothern blot using MspI and HpaII enzymes could give us some rough information, but we would like to exploit also the possibilities given by the bisulfite methods. Unfortunately, we are completely new to this technology and we would like to get some advice on how to proceed. In particular, we would like to know:
a) Which procedure would be best to try first for a inesperienced people
Is there any kit that you would recommend for this purpose?
Thanks in advance.
Hi Arturo, welcome to the forum!
As for hypomethylation analysis, there is nothing special compared to hypermethylation. hypo is a relative concept, if your gene is overexpressed in one cell line possibly through hypomethylation then you have to find another cell that doesn't overexpress this gene (thus the gene is methylated in the cell) as a reference.
I would recommend bisulfite genomic sequencing as the method for your study. It allows detailed mapping of CpGs. There are kits available for DNA modification.
Hope that helps.