Methylation of specific regions: bisulfite-sequencing at the single oocyte or 2-cell embryo level

Methods Mol Biol. 2015:1222:209-26. doi: 10.1007/978-1-4939-1594-1_16.

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.

MeSH terms

  • Animals
  • Blastocyst / cytology*
  • Blastocyst / physiology
  • DNA / isolation & purification
  • DNA Methylation*
  • Female
  • Homeodomain Proteins / genetics
  • Humans
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / genetics
  • Oocytes / physiology*
  • Polymerase Chain Reaction / methods*
  • Promoter Regions, Genetic
  • Sequence Analysis, DNA / methods*
  • Sulfites*

Substances

  • Homeodomain Proteins
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Sulfites
  • DNA
  • hydrogen sulfite