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Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection

Zinellu, Angelo and Sotgia, Salvatore and De Murtas, Valentina and Cossu Rocca, Paolo Alessandro and De Miglio, Maria Rosaria and Muroni, Maria Rosaria and Mura, Antonica and Uras, Maria Gabriela and Contini, Marcella and Deiana, Luca and Carru, Ciriaco (2011) Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection. Analytical and Bioanalytical Chemistry, Vol. 399 (3), p. 1181-1186. ISSN 1618-2642. eISSN 1618-2650. Article.

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DOI: 10.1007/s00216-010-4417-x

Abstract

Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5–1 μg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.

Item Type:Article
ID Code:6122
Status:Published
Refereed:Yes
Uncontrolled Keywords:Field-amplified sample injection, capillary electrophoresis, 5-Methylcytosine, cytosine, DNA methylation
Subjects:Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Area 06 - Scienze mediche > MED/08 Anatomia patologica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
002 Altri enti e centri di ricerca del Nord Sardegna > Porto Conte Ricerche, Alghero
001 Università di Sassari > 01 Dipartimenti > Medicina clinica, sperimentale e oncologica
002 Altri enti e centri di ricerca del Nord Sardegna > National laboratory of the national institute of biostructures and biosystems, Osilo
Publisher:Springer Berlin / Heidelberg
ISSN:1618-2642
eISSN:1618-2650
Copyright Holders:© Springer-Verlag 2010
Deposited On:08 Jun 2011 17:10

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