Cpgenome universal unmethylated dna

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Positive Control. Each run must contain two positive controls that consist of methylated DNA. The CpGenome Universal Methylated DNA (Millipore) has to be diluted to 10 ng/uL for optimal results and bisulfite converted. The Converted Methylated DNA (Promega) needs to be diluted to 3 ng/μL.

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Negative Control: Each run must contain one negative control that consists of unmethylated DNA. The CpGenome Universal Unmethylated DNA (Millipore) has to be diluted to 10 ng/μLfor optimal results and bisulfite converted.

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No Template Control (NTC). Each run must contain a no template control (NTC) that consists of all reagents with the exception of any DNA sample.

Note: The bisulfite conversion of the controls must be done at the same time as the samples.

10 nL of the resultant cleavage reactions was spotted onto silicon matrix‐preloaded chips (Spectro‐CHIP; Sequenom) using a MassARRAY nanodispenser (Sequenom) and analyzed using the MassARRAY Compact System matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometer (MALDI‐TOF) (Sequenom). The spectra's methylation ratios were calculated using epityper software v1.0 (Sequenom). The method yields quantitative results for each of the sequence‐defined analytic units referred as CpG units, which may contain either one individual CpG site or an aggregate of downstream CpG sites. Triplicate independent analyses from sodium bisulfite‐treated DNA sample were undertaken.
The effectiveness of the entire experimental procedure was assayed by analyzing as control CpGenome Universal Unmethylated DNA (Chemicon) and CpGenome Universal Methylated DNA (Chemicon, Millipore, Germany) in serial mixtures of methylated and unmethylated products, with 10% methylation increments.
Data quality control and filtering were carried out by the removal of the CpG dinucleotides whose the measurement success rate was <80%. Poor‐quality and nonvaluable data for the quantitative methylation of each CpG unit measured by MALDI‐TOF‐MS were excluded.

For quantitative methylation analysis, TaqMan methylation specific PCR (Q-MSP) was carried out using the iQ™ Multiplex Powermix (Bio-Rad) in triplicate on the iCycler iQ™ Real-Time PCR Detection system (Bio-Rad). PCR conditions and sequences are provided in S1 Table. Serial dilutions of bisulfite modified CpGenome universal methylated DNA (Chemicon International, Temecula, CA) were used to construct the calibration curve on each plate as methylation positive controls and CpGenome universal unmethylated DNA (Chemicon International) was used as the negative control. The methylation value (TaqMeth value) was defined as the ratio of methylated PGP9.5, NMDAR2B, CCNA1, or DAPK normalized to methylated β-actin, which was then multiplied by 100.

10 nl of the resultant cleavage reactions were spotted onto silicon matrix preloaded chips (Spectro-CHIP; Sequenom) using the MassARRAY nanodispenser (Sequenom) and analysed using the MassARRAY Compact System matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF) (Sequenom). The spectra’s methylation ratios were calculated using EPITYPER software v1.0 (Sequenom). The method yields quantitative results for each of the sequence-defined analytic units referred as CpG units, which may contain either one individual CpG site or an aggregate of CpG sites. Triplicate independent analyses from sodium bisulfite-treated DNA samples were undertaken. The effectiveness of the entire experimental procedure was assessed by analyzing as control CpGenome Universal Unmethylated DNA (Chemicon) and CpGenome Universal Methylated DNA (Chemicon, Millipore, Germany) in serial mixtures of methylated and unmethylated products, with 10% methylation increments. Data quality control and filtering were carried out by the removal of the CpG dinucleotides whose the measurement success rate was <90%. Poor-quality and non-valuable data for the quantitative methylation of each CpG unit measured by MALDI-TOF-MS were excluded.

Three different types of biological samples were used to validate the OPERA_MET-A panel: n.3 lung paired tumor/non-neoplastic FFPE tissues (830T/N, 881T/N, 889T/N), n.3 lung tumors optimal cutting temperature compound (OCT) embedded (435T, 475T, 495T), n.2 lung cell lines (tumor A459 and normal MRC5) were used. Tissues were collected from anonymous patients, according to the guidelines of the Local Ethical Committee of IRCCS Casa Sollievo della Sofferenza Hospital, Italy, whereas cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). A commercially available fully methylated (>95%, CpGenome Universal Methylated DNA, Millipore, Chemicon) and unmethylated (<5%, CpGenome Universal Unmethylated DNA, Chemicon) genomic gDNA with four mixtures (~25%, ~50%, ~75% and ~90%) were used as positive and negative controls to the optimal DNA conversion and library preparation.