EVALUATION OF DNA METHYLATION IN FORMALIN-FIXED, PARAFFIN- EMBEDDED COLORECTAL CANCER TISSUES (FFPE)
DOI:
https://doi.org/10.35509/01239015.704Keywords:
DNA Methylation, 5-Methylcytosine, Ras Genes, Colorectal Neoplasms, Paraffin-Embedded TissueAbstract
Alterations in methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer with potential use as a biomarker. Their evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues pose a great challenge given the partial degradation, crosslinking and low amounts of the DNA obtained. In this technical note, we describe a protocol to study the methylation status of the distal K-RAS proto-oncogene promoter from several samples obtained from two 11-year old FFPE colorectal cancer FFPE tissues. A bisulfite conversion protocol was used, alternative to the usual protocol; a modified DNA polymerase and a nested PCR were used, and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation status in other genes and types of cancer.
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