The top three hypermethylated genes were ADAM19, FLI1, and MSC, and methylation profiling was performed in these three genes using a MassARRAY EpiTYPER (Agena Bioscience, San Diego, CA, USA) based on MassCLEAVE base-specific cleavage and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Primers were designed using the Epidesigner online software, and the quantitative methylation data for each CpG site or aggregates of multiple CpG sites were obtained from MassARRAY and analyzed using EpiTYPER software (Agena Bioscience, San Diego, CA, USA). MassARRAY and NGS were used to quantify methylation states. Tumor and adjacent normal tissues were both used to perform methylation profiling, and the average methylation value of CpG sites was used to measure methylation. If the methylation value was more than the mean + 2SD of the normal control group, the case was defined as hypermethylation.
Epityper massarray
Manufactured by Agena
Sourced in United States
The EpiTYPER MassARRAY is a laboratory instrument designed for DNA methylation analysis. It utilizes the MassARRAY platform to perform quantitative assessment of DNA methylation patterns. The system combines base-specific enzymatic cleavage with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to detect and quantify DNA methylation levels.
Automatically generated - may contain errors
6 protocols using epityper massarray
1
Hypermethylation Profiling in Oncogenes
2
DNA Methylation Analysis Using MassARRAY
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methylation analysis using the MassARRAY EpiTYPER (Agena Bioscience, San Diego, CA, USA) was performed according to the standard protocol by Suchiman et al. [33 (link)]. In brief, DNA quality was examined using gel electrophoresis, and the concentration was quantified using a Quant-it PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Tokyo, Japan). Bisulfite treatment was performed with 500 ng of extracted DNA using an EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Subsequently, PCR amplification was performed in triplicate with bisulfite-treated genomic DNA using primers for each genomic locus, which were designed using the web tool EpiDesigner (https://www.epidesigner.com/ ). All the amplicon information is given in Table S2 . After T-cleavage reactions using a MassCLEAVE™ Reagent Kit (Agena Bioscience, San Diego, CA, USA), samples were transferred to a 384 SpectroCHIP and read by a mass spectrometer MassARRAY system (Agena Bioscience, San Diego, CA, USA). The DNA methylation levels for each CpG unit were calculated by EpiTYPER software v1.3 (Agena Bioscience). The data passed all the quality controls described in reference [33 (link)] and were used for the analyses in this study.
3
DNA Methylation Quantification via MassARRAY
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNAm signal validation and replication was done by the EpiTYPER MassARRAY assay (Agena Bioscience). This assay uses a MALDI-TOF mass spectrometry-based method to quantitatively assess the DNA methylation state of the CpG sites of interest [53 (link)]. DNA (500 ng) was bisulfite-converted as indicated in Section 4.3 .
PCR amplification, treatment with SAP solution and Transcription/RNase A cocktails were performed according to the manufacturer’s instructions, and the mass spectra were analyzed by an EpiTYPER analyzer. The MassARRAY assay cannot discriminate between CpGs located in close proximity in the sequence, so instead, the close neighboring CpGs are analyzed as “Units”, i.e., the measured methylation level is the average of the methylation levels of the CpGs cumulatively analyzed within the Unit. In the case of cg03546163, the measured methylation level is the average between two CpG sites located in very close proximity (Figure S1 ). For cg06633438, the two adjacent signals were considered, since the results for the model did not differ for effect size, standard error, 95% CI or p value (Figure S2 ).
PCR amplification, treatment with SAP solution and Transcription/RNase A cocktails were performed according to the manufacturer’s instructions, and the mass spectra were analyzed by an EpiTYPER analyzer. The MassARRAY assay cannot discriminate between CpGs located in close proximity in the sequence, so instead, the close neighboring CpGs are analyzed as “Units”, i.e., the measured methylation level is the average of the methylation levels of the CpGs cumulatively analyzed within the Unit. In the case of cg03546163, the measured methylation level is the average between two CpG sites located in very close proximity (
4
Analysis of ZBTB12 DNA Methylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Buffy coats of peripheral blood cells were isolated from whole blood samples collected in sodium citrate EDTA tubes and centrifuged at 3000 rpm for 20 min at RT. DNA extraction was done using a silica matrix-based method as described [29 ]. Of the 754 Moli-family participants, 623 had good quality DNA samples to perform the methylation analysis. We measured ZBTB12 methylation using the Sequenom EpiTYPER MassARRAY (Agena) platform [15 (link)]. Details of the ZBTB12 region studied (chr6: 31899847-31900326, GRCh38/hg38 Assembly) are reported by Guarrera and colleagues [5 (link)]. Bisulfite treatment was conducted on 1 μg of genomic DNA using the MethylDetector kit (Active Motif) as described [15 (link)]. All PCR amplifications were performed in duplicate. For the CpG-specific analysis, data were discarded when the duplicate measurements had a standard deviation (SD) ≥ 5% [15 (link), 30 (link), 31 (link)]. Sequenom peaks with reference intensity > 2 and overlapping units were excluded from the analysis [15 (link), 30 (link), 31 (link)]. To exclude possible intra-plate differences, a sample of K562 DNA was carried on in each plate as an internal control.
Of the 20 CpG units included in the ZBTB12 region studied [5 (link)] (CGI1 in Fig.1 ), we excluded the ones having more than 15% of missing values in the Moli-family cohort, leading to a total of 13 CpG (Table 1 ).
Of the 20 CpG units included in the ZBTB12 region studied [5 (link)] (CGI1 in Fig.
5
Methylation Analysis of PEAR1 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PEAR1 (CGI1) methylation was evaluated using the Sequenom EpiTYPER MassARRAY (Agena) platform as described [30 (link), 40 (link), 41 (link)] on white blood cell DNA from 605 Moli-family and 1002 FLEMENGHO participants.
Bisulfite treatment was conducted on 1 μg of genomic DNA using the MethylDetector kit (Active Motif) according to the manufacturer’s instructions, except for the incubation protocol during the conversion, performed for a total of 16 h as described [70 (link)]. The amplicon to study PEAR1 methylation was designed using the Sequenom EpiDesigner software (http://www.epidesigner.com/ ) [30 (link)]. All PCR amplifications were performed in duplicate. For the CpG-specific analysis, data were discarded when the duplicate measurements had a SD equal to or greater than 5% [30 (link), 40 (link), 41 (link)]. Sequenom peaks with reference intensity above 2 and overlapping units were excluded from the analysis [30 (link), 40 (link), 41 (link)]. To exclude possible intra-plate differences, a sample of K562 DNA, with known PEAR1 methylation profile (around 90%), was carried on in each plate.
Bisulfite treatment was conducted on 1 μg of genomic DNA using the MethylDetector kit (Active Motif) according to the manufacturer’s instructions, except for the incubation protocol during the conversion, performed for a total of 16 h as described [70 (link)]. The amplicon to study PEAR1 methylation was designed using the Sequenom EpiDesigner software (
6
BRCA1 Methylation Predictive Biomarker
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary endpoint of the study was the evaluation of the robustness of BRCA1 protein expression assessed by IHC to predict BRCA1 promoter methylation status, assessed by the methylation-specific polymerase chain reaction (MS-PCR) assay. The key secondary endpoint was the evaluation of the concordance between the determination of BRCA1 promoter methylation status by the quantitative EpiTyper® MassARRAY® (Agena Bioscience, Sans Diego, CA, USA) and by MS-PCR. Other secondary endpoints were the evaluation of the association between BRCA1 promoter methylation status assessed by MS-PCR and clinicopathological variables, and the evaluation of the impact of these variables on overall survival (OS) and relapse-free survival (RFS).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!