ArticleBMC cancer2015
An improved sequencing-based strategy to estimate locus-specific DNA methylation.
Article in BMC cancer, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 9 citations in OpenAlex.
- Shaping Precision Medicine: The Journey of Sequencing Technologies Across Human Solid Tumors.Biomedicines · 2025Review
- Development and Clinical Validation of Molecular Subgrouping in Medulloblastoma by Targeted Methylation Sequencing.Asian Pacific journal of cancer prevention : APJCP · 2025Article
- Neural crest E-cadherin loss drives cleft lip/palate by epigenetic modulation via pro-inflammatory gene-environment interaction.Nature communications · 2023Article
- DNA Methylation in Solid Tumors: Functions and Methods of Detection.International journal of molecular sciences · 2021Review
- A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness.Cell death and differentiation · 2018Article
- "Squirrel" Primer-Based PCR Assay for Direct and Targeted Sanger Sequencing of Short Genomic Segments.Journal of biomolecular techniques : JBT · 2017Article
- Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers.Breast cancer research : BCR · 2016Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundDNA 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.
methodsOur 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.
resultsNBSP 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.
conclusionsNBSP 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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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.