title: Methylation of specific regions: bisulfite-sequencing at the single oocyte or 2-cell embryo level.
abstract: To question the possible implication of an alteration of the DNA methylation of imprinted genes in normal development failure observed following fertilization in ART centers, it has been necessary to develop a reproducible and highly efficient method to perform analysis at the one cell level. We have thus developed a very efficient protocol for methylation studies on individual oocytes or cleavage-stage embryos. All the different steps were optimized, from DNA extraction, to limit DNA degradation and give a high success rate of bisulfite converted DNA, to amplification of the bisulfite modified DNA.
authors.affiliation: Institut Cellule Souche et Cerveau, INSERM U846, 18 Av Doyen Lépine, 69500, Bron, France.
authors.forename: Annick
authors.initials: A
authors.lastname: Lefèvre
authors.forename: Thierry
authors.initials: T
authors.lastname: Blachère
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D004247:DNA / Q000302:isolation & purification; D019175:DNA Methylation; D005260:Female; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D009865:Oocytes / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite