title: Correction of PCR-bias in quantitative DNA methylation studies by means of cubic polynomial regression.
abstract: DNA methylation profiling has become an important aspect of biomedical molecular analysis. Polymerase chain reaction (PCR) amplification of bisulphite-treated DNA is a processing step that is common to many currently used methods of quantitative methylation analysis. Preferential amplification of unmethylated alleles-known as PCR-bias-may significantly affect the accuracy of quantification. To date, no universal experimental approach has been reported to overcome the problem. This study presents an effective method of correcting biased methylation data. The procedure includes a calibration performed in parallel to the analysis of the samples under investigation. DNA samples with defined degrees of methylation are analysed. The observed deviation of the experimental results from the expected values is used for calculating a regression curve. The equation of the best-fitting curve is then used for correction of the data obtained from the samples of interest. The process can be applied irrespective of the locus interrogated and the number of sites analysed, avoiding an optimization of the amplification conditions for each individual locus.
authors.affiliation: Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. e.moskalev@dkfz-heidelberg.de
authors.forename: Evgeny A
authors.initials: EA
authors.lastname: Moskalev
authors.forename: Mikhail G
authors.initials: MG
authors.lastname: Zavgorodnij
authors.forename: Svetlana P
authors.initials: SP
authors.lastname: Majorova
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Vorobjev
authors.forename: Pouria
authors.initials: P
authors.lastname: Jandaghi
authors.forename: Irina V
authors.initials: IV
authors.lastname: Bure
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Hoheisel
mesh_terms: D000483:Alleles; D001402:B-Lymphocytes / Q000378:metabolism; D002138:Calibration; D045744:Cell Line, Tumor; D019175:DNA Methylation; D006801:Humans; D007938:Leukemia / Q000235:genetics; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results