Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP

Details

Serval ID
serval:BIB_22AAE54DFB3C
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP
Journal
BioTechniques
Author(s)
Bian  Y. S., Yan  P., Osterheld  M. C., Fontolliet  C., Benhattar  J.
ISSN
0736-6205 (Print)
Publication state
Published
Issued date
2001
Volume
30
Number
1
Pages
66-72
Notes
PT - Research Support, Non-U.S. Gov't PT - Technical Report
Abstract
Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified
Keywords
Dna/genetics/metabolism/DNA Methylation/Dissection/Female/Humans/Neoplasms/Pathology/Paraffin Embedding/Placenta/Polymerase Chain Reaction/methods/Polymorphism,Single-Stranded Conformational/Promoter Regions (Genetics)/Reproducibility of Results/Sensitivity and Specificity/Sulfites/Tissue Fixation/Tumor Cells,Cultured
Pubmed
Web of science
Create date
29/01/2008 18:35
Last modification date
20/08/2019 13:00
Usage data