Rapid and quantitative detection of CpG-methylation status using TaqMan PCR combined with methyl-binding-domain polypeptide.
OBJECTIVE
To assess the medical applicability of CpG methylation as molecular markers for cancer diagnosis, we established a new system to determine DNA methylation based on TaqMan PCR combined with a methyl-binding-domain polypeptide 2.


METHODS
We evaluated the diagnostic applicability of this approach by examining the methylation status of two tumor suppressor genes, RASSF1A and APC, in 10 paired hepatocellular carcinoma (HCC) and the corresponding non-tumor liver tissues.


RESULTS
Methylation levels of total 20 clinical samples measured by the TaqMan PCR assay showed a significantly positive correlation (R=0.814, P<0.0005 for RASSF1A, R=0.736, P<0.00001 for APC) with those calculated by bisulfite sequencing. The methylated DNA amount measured by our TaqMan PCR system precisely replicated the methylation status estimated by direct sequencing.


CONCLUSIONS
This suggests our method may serve as a reliable and easy-to-use tool for cancer diagnosis using methylated genes as biomarkers.
Kimura
Naoki
N
Research Group, Molecular Diagnostics R&D Department, Roche Diagnostics K.K., 6-1, Shiba 2-chome, Minato-ku, Tokyo 105-0014, Japan.
Moribe
Toyoki
T
Iizuka
Norio
N
Miura
Toshiaki
T
Tamatsukuri
Shigeru
S
Ishitsuka
Hideo
H
Hamamoto
Yoshihiko
Y
Oka
Masaaki
M