title: A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction.
abstract: DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy.
authors: Department of Biology and Genetics, Medical University of Gdańsk, 80-210 Gdańsk, Poland.
authors: Magdalena
authors: M
authors: Krygier
authors: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, 80-233 Gdańsk, Poland.
authors: Justyna
authors: J
authors: Podolak-Popinigis
authors: Department of Biology and Genetics, Medical University of Gdańsk, 80-210 Gdańsk, Poland.
authors: Janusz
authors: J
authors: Limon
authors: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, 80-233 Gdańsk, Poland.
authors: Paweł
authors: P
authors: Sachadyn
authors: Department of Biology and Genetics, Medical University of Gdańsk, 80-210 Gdańsk, Poland. Electronic address: astanisl@gumed.edu.pl.
authors: Anna
authors: A
authors: Stanisławska-Sachadyn
mesh_terms: D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004262:DNA Restriction Enzymes
keywords: Assay; MSRE–qPCR; Methylation; Methylation-sensitive restriction enzyme; Validation