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BMC Cancer
Published in: BMC Cancer 1/2015

Open Access 01-12-2015 | Technical advance

An improved sequencing-based strategy to estimate locus-specific DNA methylation

Authors: Giulia Brisotto, Alessandra di Gennaro, Valentina Damiano, Michela Armellin, Tiziana Perin, Roberta Maestro, Manuela Santarosa

Published in: BMC Cancer | Issue 1/2015

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Abstract

Background

DNA methylation is an important epigenetic mechanism of transcriptional control that plays an essential role in several cellular functions. Aberrant DNA methylation in cancer has been frequently associated with downregulation of microRNAs and protein coding genes, such as miR-200c/miR-141 cluster and E-cadherin. Current strategies to assess DNA methylation, including bisulfite treatment-based assays, tend to be time-consuming and may be quite expensive when a precise appraisal is required. The Sanger-sequencing of the amplified bisulfite-treated DNA (BSP) might represent a practical option to measure DNA methylation at single CpG resolution. However, this strategy often produces noisy data, which affects accurate quantification. Here we propose an improved, reliable and cost-effective BSP-based protocol that allows proper DNA methylation assessment.

Methods

Our strategy, named normalized-BSP (NBSP), takes advantage of tailed C-balanced primers and a normalization procedure based on C/T ratio to overcome BSP-associated noise problems and nucleotide signal unbalance. NBSP was applied to estimate miR-200c/miR-141 locus methylation in serial dilution experiments and was compared to conventional methods. Besides, it was applied in the analysis of FFPE breast cancer samples and further validated in the context of the E-cadherin promoter.

Results

NBSP strategy outperformed conventional BSP in the estimate of the fraction of methylated cytosine in serial dilution experiments, providing data in agreement with the widely used but cumbersome cloning-based protocol. This held true for both miR-200c/miR-141 locus and E-cadherin promoter analyses. Moreover, the miR-200c/miR-141 locus methylation reflected the decrease in miRNA expression both in breast cancer cell lines and in the FFPE samples.

Conclusions

NBSP is a rapid and economical method to estimate the extent of methylation at each CpG of a given locus. Notably, NBSP works efficiently on FFPE samples, thus disclosing the perspective of its application also in the diagnostic setting.
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Metadata
Title
An improved sequencing-based strategy to estimate locus-specific DNA methylation
Authors
Giulia Brisotto
Alessandra di Gennaro
Valentina Damiano
Michela Armellin
Tiziana Perin
Roberta Maestro
Manuela Santarosa
Publication date
01-12-2015
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2015
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-015-1646-6

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