Methylationsensitive restriction enzymedroplet digital PCR assay for the onestep highly sensitive analysis of DNA methylation hotspots.
Int J Mol Med
; 53(5)2024 05.
Article
en En
| MEDLINE
| ID: mdl-38488030
ABSTRACT
DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the highthroughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the MethylationSensitive Restriction Enzymedroplet digital PCR (MSREddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the highsensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSREddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSREddPCR paving the way to the analysis of other methDNA hotspots in different tumors.
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Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Sulfitos
/
Metilación de ADN
/
Melanoma
Límite:
Humans
Idioma:
En
Año:
2024
Tipo del documento:
Article