Massarray epityper system

Ten amplicons that were shown to be hypermethylated in UCa cases and whose genomic coordinates and primer sequences have been described previously [12 (link)] were analyzed by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry (MassARRAY EpiTYPER system, Agena Bioscience GmbH, Hamburg, Germany), enabling the quantitative measurement of CpG methylation at single dinucleotide resolution [40 (link),41 (link)]. For this purpose, 250 ng of DNA were bisulfite-converted using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s protocol. All necessary preparations preceding mass spectrometry (PCR, reverse transcription and RNA cleavage) and the final MALDI-TOF mass spectrometry was performed as previously described [12 (link)]. When neighboring CpGs were located on identical mass fragments and could not be resolved at the individual level, a comprehensive DNA methylation value for the whole “CpG unit” was obtained. CpGs with ambiguous mass results, e.g., due to disturbing signals resulting from CpG-free masses, have been excluded from the 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)].

Genomic DNA (500 ng) was bisulfite treated using the EZ-DNA methylation kit (Zymo, Irvine, CA). DNA sequence of interest was amplified using PCR with the bi-sulfite treated DNA as the template and primers that incorporate the T7 tag (28 (link)). Sequences of interest were located in the promoter region of Srebf1 and Lep, and differentially methylated region 0 (DMR0) of Igf2 (29 (link)). Primer information was included in Supplementary Table 1. The amplicons were analyzed with the MassArray EpiTyper system (Agena Biosciences, San Diego, CA, USA) at the Epigenetic core facility of City University of New York (CUNY) Advanced Science Research Center (ASRC). This system uses matrix-assisted laser desorption ionization/time of flight mass spectrometry to assess methylation of each CpG unit within the amplicon. Each CpG unit may contain one or more adjacent CpG dinucleotides that cannot be resolved individually. The CpG sites and unit information of each sequence measured were provided in Supplementary Figure 2. Only CpG units with over 75% of success rate across samples were included in the final analysis.