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Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection

Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and repro...

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Autores principales: Zinellu, Angelo, Sotgiu, Elisabetta, Assaretti, Stefano, Sotgia, Salvatore, Paliogiannis, Panagiotis, Pintus, Gianfranco, Mangoni, Arduino A., Carru, Ciriaco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733158/
https://www.ncbi.nlm.nih.gov/pubmed/29333318
http://dx.doi.org/10.1155/2017/4065892
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author Zinellu, Angelo
Sotgiu, Elisabetta
Assaretti, Stefano
Sotgia, Salvatore
Paliogiannis, Panagiotis
Pintus, Gianfranco
Mangoni, Arduino A.
Carru, Ciriaco
author_facet Zinellu, Angelo
Sotgiu, Elisabetta
Assaretti, Stefano
Sotgia, Salvatore
Paliogiannis, Panagiotis
Pintus, Gianfranco
Mangoni, Arduino A.
Carru, Ciriaco
author_sort Zinellu, Angelo
collection PubMed
description Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, v/v) allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA.
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spelling pubmed-57331582018-01-14 Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection Zinellu, Angelo Sotgiu, Elisabetta Assaretti, Stefano Sotgia, Salvatore Paliogiannis, Panagiotis Pintus, Gianfranco Mangoni, Arduino A. Carru, Ciriaco J Anal Methods Chem Research Article Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, v/v) allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA. Hindawi 2017 2017-12-03 /pmc/articles/PMC5733158/ /pubmed/29333318 http://dx.doi.org/10.1155/2017/4065892 Text en Copyright © 2017 Angelo Zinellu et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Zinellu, Angelo
Sotgiu, Elisabetta
Assaretti, Stefano
Sotgia, Salvatore
Paliogiannis, Panagiotis
Pintus, Gianfranco
Mangoni, Arduino A.
Carru, Ciriaco
Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection
title Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection
title_full Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection
title_fullStr Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection
title_full_unstemmed Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection
title_short Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection
title_sort evaluation of global genomic dna methylation in human whole blood by capillary electrophoresis uv detection
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733158/
https://www.ncbi.nlm.nih.gov/pubmed/29333318
http://dx.doi.org/10.1155/2017/4065892
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