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