title: Methylation-specific ligation detection reaction (msLDR): a new approach for multiplex evaluation of methylation patterns.
abstract: A new sensitive method for multiplex gene-specific methylation analysis was developed using a ligation-based approach combined with a TaqMan-based detection and readout employing universal reporter probes. The approach, termed methylation-specific Ligation Detection Reaction (msLDR), was applied to test 16 loci in 8 different colorectal cancer cells in parallel. These loci encode immune regulatory genes involved in T-cell and natural killer cell activation, whose silencing is associated with the development or progression of colorectal cancer. Parallel analysis of HLA-A, HLA-B, STAT1, B2M, LMP2, LMP7, PA28α, TAP1, TAP2, TAPBP, ULBP2 and ULBP3 by msLDR in eight colorectal cancer cell lines showed preferential methylation at the HLA-B, ULBP2 and ULBB3 loci, but not at the other loci. MsLDR was found to represent a suitable and sensitive method for the detection of distinct methylation patterns as validated by conventional bisulphite Sanger sequencing and COBRA analysis. Since gene silencing by epigenetic mechanisms plays a central role during transformation of a normal differentiated somatic cell into a cancer cell, characterization of the gene methylation status in tumours is a major topic not only in basic research, but also in clinical diagnostics. Due to a very simple workflow, msLDR is likely to be applicable to clinical samples and thus comprises a potential diagnostic tool for clinical purposes.
authors: Institute of Pathology, University Medicine Charité, Charitéplatz1, 10117 Berlin, Germany. felix.bormann@charite.de
authors: Felix
authors: F
authors: Bormann
authors: Christine
authors: C
authors: Sers
authors: Barbara
authors: B
authors: Seliger
authors: Diana
authors: D
authors: Handke
authors: Thomas
authors: T
authors: Bergmann
authors: Stephanie
authors: S
authors: Seibt
authors: Hans
authors: H
authors: Lehrach
authors: Andreas
authors: A
authors: Dahl
mesh_terms: D017951:Antigen Presentation / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D058851:GPI-Linked Proteins / Q000235:genetics; D005805:Genes, MHC Class I; D005821:Genetic Techniques / Q000706:statistics & numerical data; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D007694:Killer Cells, Natural / Q000276:immunology; D008904:Miniaturization; D016133:Polymerase Chain Reaction / Q000379:methods; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D004273:DNA, Neoplasm; D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; C423230:ULBP2 protein, human; C423229:ULBP3 protein, human