title: Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection.
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.
authors.affiliation: Porto Conte Ricerche Srl, Tramariglio, 07041 Alghero, Sassari, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Zinellu
authors.forename: Salvatore
authors.initials: S
authors.lastname: Sotgia
authors.forename: Valentina
authors.initials: V
authors.lastname: De Murtas
authors.forename: Paolo
authors.initials: P
authors.lastname: Cossu-Rocca
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: De Miglio
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Muroni
authors.forename: Antonica
authors.initials: A
authors.lastname: Mura
authors.forename: Maria Gabriela
authors.initials: MG
authors.lastname: Uras
authors.forename: Marcella
authors.initials: M
authors.lastname: Contini
authors.forename: Luca
authors.initials: L
authors.lastname: Deiana
authors.forename: Ciriaco
authors.initials: C
authors.lastname: Carru
mesh_terms: D002138:Calibration; D015179:Colorectal Neoplasms / Q000378:metabolism*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008223:Lymphoma / Q000378:metabolism*; D008745:Methylation; D016612:Paraffin Embedding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde