scholarly journals Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application

2014 ◽  
Vol 2 (1) ◽  
pp. 28-36 ◽  
Author(s):  
Benedetta Izzi ◽  
Alexandra M. Binder ◽  
Karin B. Michels
Keyword(s):  
Bisulfite Conversion

scholarly journals Methods for CpG Methylation Array Profiling Via Bisulfite Conversion

2018 ◽  
pp. 233-254 ◽  
Author(s):  
Fatjon Leti ◽  
Lorida Llaci ◽  
Ivana Malenica ◽  
Johanna K. DiStefano
Keyword(s):  
Cpg Methylation ◽  
Bisulfite Conversion ◽  
Methylation Array

Bisulfite Conversion

2013 ◽  
pp. 149-150
Author(s):  
Vani Brahmachari ◽  
Shruti Jain
Keyword(s):  
Bisulfite Conversion

scholarly journals Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients

2020 ◽  
Vol 21 (4) ◽  
pp. 1547 ◽  
Author(s):  
Elisa Boldrin ◽  
Matteo Curtarello ◽  
Marco Dallan ◽  
Rita Alfieri ◽  
Stefano Realdon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Esophageal Adenocarcinoma ◽  
Methodological Approach ◽  
Methylation Status ◽  
Dna Amplification ◽  
Restriction Enzymes ◽  
Poor Quality ◽  
Bisulfite Conversion ◽  
Dna Hypomethylation ◽  
Methylation Patterns

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett’s esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

Influence of DNA Sequence and Methylation Status on Bisulfite Conversion of Cytosine Residues

1995 ◽  
Vol 231 (1) ◽  
pp. 263-265 ◽  
Author(s):  
K.I. Rother ◽  
J. Silke ◽  
O. Georgiev ◽  
W. Schaffner ◽  
K. Matsuo
Keyword(s):  
Dna Sequence ◽  
Methylation Status ◽  
Bisulfite Conversion

scholarly journals Comparison of enzymatic-and bisulfite conversion to map the plasma cell-free methylome in cancer

2019 ◽  
Vol 30 ◽  
pp. v13
Author(s):  
N. Lambert ◽  
A. Robertson ◽  
R. Srivas ◽  
N. Peterman ◽  
J. Close ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Bisulfite Conversion

scholarly journals DNA Methylation Validation Methods: a Coherent Review with Practical Comparison

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Šárka Šestáková ◽  
Cyril Šálek ◽  
Hana Remešová
Keyword(s):  
Dna Methylation ◽  
Accurate Method ◽  
Primer Design ◽  
Clinical Diagnostics ◽  
Bisulfite Conversion ◽  
Design And Optimization ◽  
Validation Methods ◽  
Specific Restriction ◽  
Polymerase Chain ◽  
Pcr Conditions

Abstract Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods’ feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

Screening and Triage Test for Early Detection of Gastric Cancer (STEAD-GC): A noninvasive trial for early detection of gastric cancer.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 17-17
Author(s):  
Alejandro Corvalan ◽  
Maria Jose Maturana ◽  
Marianela Sanchez ◽  
Alfonso Calvo ◽  
Catterina Ferreccio
Keyword(s):  
Gastric Cancer ◽  
Early Detection ◽  
Dna Extraction ◽  
Mass Screening ◽  
Chronic Gastritis ◽  
Plasma Samples ◽  
Bisulfite Conversion ◽  
Non Invasive ◽  
Tumor Tissues ◽  
Triage Test

17 Background: Gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. Previously, we identified a potential biomarker for non-invasive detection of GC, the DNA methylation of the promoter region of Reprimo, a p53-dependent G2 arrest mediator candidate (Clin Cancer Res 2008;14:6264-9). Furthermore, we developed a quantitative assay (MethyLight) for a mass screening of GC (DDW2011-1029128). Here we reported the preliminary findings of our ongoing prospective trial STEAD-GC (Screening and Triage test for Early Detection of Gastric Cancer) which is being conducted in Chile, a country with a high mortality rate for GC. Methods: Twenty GC cases (tumor, non-tumor tissues and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. Results: Concentrations of DNA were similar in both groups (Avg 32.2 ng/ml [range: 8.9-70.8 ng/ml]). The average DNA levels of Reprimo were higher in GC [964,215 copies/ml, 539,593 copies/ml and 80,113 copies/ml in tumor, non-tumor and plasma, respectively] but lower in symptomatic chronic gastritis [137,721 copies/ml and 8,387 copies/ml, tissue and plasma, respectively]. Methods: Twenty GC cases (tumor, non-tumor tissues, and plasma samples) and 41 symptomatic chronic gastritis cases (29 tissues and 12 pairs of tissue and plasma samples) were evaluated for Reprimo levels by MethyLight after DNA extraction and bisulfite conversion. Conclusions: By using our previous cut-off of 15,125 copies/ml, our method correctly identified 10 out of 12 gastritis cases and 16 out of 20 GC cases (p value <0.001). Our data confirms our non-invasive method for early detection of GC may be suitable for a mass screening of GC.

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