LUMA (LUminometric Methylation Assay)--a high throughput method to the analysis of genomic DNA methylation.
Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200-500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer.
Karimi
Mohsen
M
Departments of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Johansson
Sofia
S
Stach
Dirk
D
Corcoran
Martin
M
Grandér
Dan
D
Schalling
Martin
M
Bakalkin
Georgy
G
Lyko
Frank
F
Larsson
Catharina
C
Ekström
Tomas J
TJ
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D005821:Genetic Techniques; D016678:Genome / Q000502:physiology*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice
D004247:DNA; D004262:DNA Restriction Enzymes; D001374:Azacitidine