Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction.
We have developed a quantitative method for methylation analysis of the p16 gene based on real-time methylation-specific PCR (MSP). Real-time MSP is sensitive enough to detect down to 10 genome equivalents of the methylated p16 sequence. Application of real-time MSP to DNA from tumor-derived cell lines revealed complete concordance with conventional MSP analysis. Quantitative data generated by real-time MSP were expressed as the methylation index, which was defined as the percentage of bisulfite-converted DNA that consisted of methylated target sequences. The methylation index was shown to be inversely correlated with p16 gene transcription during demethylation treatment of cell lines with 5-aza-2'-deoxycytidine. The application of real-time MSP to bone marrow aspirates from patients with multiple myeloma revealed complete concordance with conventional MSP analysis. Real-time quantitative MSP may have applications in elucidating diverse biological processes involving DNA methylation and may become a valuable diagnostic tool for detecting tumor-associated epigenetic changes in cancer patients.
Lo
Y M
YM
Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories. loym@cuhk.edu.hk
Wong
I H
IH
Zhang
J
J
Tein
M S
MS
Ng
M H
MH
Hjelm
N M
NM
D019175:DNA Methylation; D019942:Genes, p16; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors