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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

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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.

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Abbreviations

BGE:

background electrolyte

CE:

capillary electrophoresis

FASI:

field-amplified sample injection

FFPE:

formalin-fixed paraffin-embedded

HPLC:

high-performance liquid chromatography

Tris:

tris(hydroxymethyl)aminomethane

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Acknowledgements

This study was supported by the Fondazione Banco di Sardegna, Sassari, Italy, and by the Ministero dell’Università e della Ricerca, Italy. A.Z. was supported by Regione Autonoma della Sardegna Ricerca cofinanziata Programma Operativo FSE Sardegna 2007-2013 - L.R.7/2007 - Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna. The manuscript language revision by Maria Antonietta Meloni is greatly appreciated.

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Authors and Affiliations

  1. Porto Conte Ricerche Srl, Tramariglio, 07041, Alghero, Sassari, Italy

    Angelo Zinellu

  2. Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100, Sassari, Italy

    Angelo Zinellu, Salvatore Sotgia, Valentina De Murtas, Maria Rosaria De Miglio, Maria Rosaria Muroni, Luca Deiana & Ciriaco Carru

  3. Department of Surgical Pathology, University of Sassari, 07100, Sassari, Italy

    Paolo Cossu-Rocca, Antonica Mura, Maria Gabriela Uras & Marcella Contini

  4. National Laboratory of the National Institute of Biostructures and Biosystems, 07033, Osilo, Italy

    Ciriaco Carru

Authors
  1. Angelo Zinellu
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  2. Salvatore Sotgia
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  3. Valentina De Murtas
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  4. Paolo Cossu-Rocca
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  5. Maria Rosaria De Miglio
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  6. Maria Rosaria Muroni
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  7. Antonica Mura
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  8. Maria Gabriela Uras
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  9. Marcella Contini
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  10. Luca Deiana
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  11. Ciriaco Carru
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Correspondence to Ciriaco Carru.

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Zinellu, A., Sotgia, S., De Murtas, V. et al. Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection. Anal Bioanal Chem 399, 1181–1186 (2011). https://doi.org/10.1007/s00216-010-4417-x

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  • DOI: https://doi.org/10.1007/s00216-010-4417-x

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