Quantitative DNA Methylation Analysis

The potentially bladder specific target region deduced from the comparison of the screening experiments in cell culture and urine was assessed in more detail by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry (MassARRAY EpiTYPER system, Agena Bioscience GmbH, Hamburg, Germany) which enables the quantitative measurement of CpG methylation at single dinucleotide resolution [22 (link),23 (link)]. For this purpose, primers covering a DNA-stretch which includes the two most promising candidate sites were designed by Agena`s EpiDesigner software (http://www.epidesigner.com/index.html). The sequences were aggaagagagGGGTTATGTTGAGAAGTAAGGAATGT (forward-primer) and cagtaatacgactcactatagggagaaggctCCCACACAAAACTTAAAAATAAAACTT (reverse primer, the small print represents the respective tags required by the method). The amplified region was CHR6:28911328-28911620 (genome build GRCh37/hg19). All analyses were carried out according to the protocol of Agena Bioscience GmbH and have been previously described in detail [15 (link)].

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Köhler C.U., Walter M., Lang K., Plöttner S., Roghmann F., Noldus J., Tannapfel A., Tam Y.C., Käfferlein H.U, & Brüning T. (2020). In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer. Biomedicines, 8(8), 233.

Publication 2020

MassARRAY EpiTYPER system EpiDesigner software

A dna Bladder Cell culture Dinucleotide Genome Maldi Mass spectrometry Methylation Primer Urine

Corresponding Organization : Institute for Prevention and Occupational Medicine

Other organizations : University of Tübingen, Universitätsklinik Marien Hospital Herne, Ruhr University Bochum, Technische Hochschule Georg Agricola

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Variable analysis

independent variables

Methylation levels of the target region

dependent variables

Quantitative measurement of CpG methylation at single dinucleotide resolution

control variables

Experimental protocol and methods as described in the cited references [15, 22, 23]

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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