title: Evaluating the methylation status of CXCR4 promoter: A cost-effective and sensitive two-step PCR method.
abstract: The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region.
authors.affiliation: Centro Cardiologico Monzino, IRCCS, Milano, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Bianchessi
authors.affiliation: Axxam SpA, Molecular Biology Unit, via Meucci 3 - 20091 Bresso, Milano, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Lauri
authors.affiliation: Centro Cardiologico Monzino, IRCCS, Milano, Italy.
authors.forename: Vera
authors.initials: V
authors.lastname: Vigorelli
authors.affiliation: Centro Cardiologico Monzino, IRCCS, Milano, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Toia
authors.affiliation: Centro Cardiologico Monzino, IRCCS, Milano, Italy. Electronic address: cristina.vinci@ccfm.it.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Vinci
mesh_terms: D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D019718:Receptors, CXCR4 / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: C527276:CXCR4 protein, human; D017931:DNA Primers; D019718:Receptors, CXCR4
keywords: CXCR4; DNA methylation; Epigenetics; Promoter; SYBR green; qPCR