title: An improved sequencing-based strategy to estimate locus-specific DNA methylation.
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.
authors.affiliation: Experimental Oncology 1, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. gbrisotto@cro.it.
authors.forename: Giulia
authors.initials: G
authors.lastname: Brisotto
authors.affiliation: Experimental Oncology 1, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. adigennaro@cro.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: di Gennaro
authors.affiliation: Experimental Oncology 1, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. vdamiano@cro.it.
authors.forename: Valentina
authors.initials: V
authors.lastname: Damiano
authors.affiliation: Experimental Oncology 1, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. marmellin@cro.it.
authors.forename: Michela
authors.initials: M
authors.lastname: Armellin
authors.affiliation: Pathology, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. tperin@cro.it.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Perin
authors.affiliation: Experimental Oncology 1, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. rmaestro@cro.it.
authors.forename: Roberta
authors.initials: R
authors.lastname: Maestro
authors.affiliation: Experimental Oncology 1, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy. msantarosa@cro.it.
authors.forename: Manuela
authors.initials: M
authors.lastname: Santarosa
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D019175:DNA Methylation; D005260:Female; D056426:Genetic Loci; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D035683:MicroRNAs