Open Access

Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots

  • Authors:
    • Giuseppe Gattuso
    • Alessandro Lavoro
    • Rosario Caltabiano
    • Gabriele Madonna
    • Mariaelena Capone
    • Paolo Antonio Ascierto
    • Luca Falzone
    • Massimo Libra
    • Saverio Candido
  • View Affiliations

  • Published online on: March 14, 2024     https://doi.org/10.3892/ijmm.2024.5366
  • Article Number: 42
  • Copyright: © Gattuso et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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 high‑throughput 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 Methylation‑Sensitive Restriction Enzyme‑droplet digital PCR (MSRE‑ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high‑sensitivity 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 MSRE‑ddPCR 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 MSRE‑ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.
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May-2024
Volume 53 Issue 5

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Spandidos Publications style
Gattuso G, Lavoro A, Caltabiano R, Madonna G, Capone M, Ascierto PA, Falzone L, Libra M and Candido S: Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots. Int J Mol Med 53: 42, 2024.
APA
Gattuso, G., Lavoro, A., Caltabiano, R., Madonna, G., Capone, M., Ascierto, P.A. ... Candido, S. (2024). Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots. International Journal of Molecular Medicine, 53, 42. https://doi.org/10.3892/ijmm.2024.5366
MLA
Gattuso, G., Lavoro, A., Caltabiano, R., Madonna, G., Capone, M., Ascierto, P. A., Falzone, L., Libra, M., Candido, S."Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots". International Journal of Molecular Medicine 53.5 (2024): 42.
Chicago
Gattuso, G., Lavoro, A., Caltabiano, R., Madonna, G., Capone, M., Ascierto, P. A., Falzone, L., Libra, M., Candido, S."Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots". International Journal of Molecular Medicine 53, no. 5 (2024): 42. https://doi.org/10.3892/ijmm.2024.5366