TY - CHAP
T1 - Analysis of DNA methylation patterns in single blastocysts by Pyrosequencing(®).
AU - Huntriss, John
AU - Woodfine, Kathryn
AU - Huddleston, Joanna E.
AU - Murrell, Adele
AU - Picton, Helen M.
PY - 2015
Y1 - 2015
N2 - Extensive epigenetic reprogramming occurs during mammalian gametogenesis and preimplantation development. DNA methylation patterns that are laid down during these stages are essential for subsequent normal foetal development. The requirement for more precise assessment of the epigenetic programming of in vitro-derived human preimplantation embryo has become of paramount importance following the identification of epigenetic diseases that are associated with assisted reproduction and/or infertility. Such techniques are also useful and applicable to experimental reproductive biology. In order to expand our knowledge of epigenetic marks, including DNA methylation, during mammalian reproduction and early development, it is necessary to test new and sufficiently sensitive protocols. There are, however, unique challenges to obtain DNA methylation data from the small cell numbers that are present in the preimplantation embryo. In this protocol, we describe the successful application of Pyrosequencing(®) to yield quantitative DNA methylation data over several CpG sites at differentially methylated regions (DMRs) at imprinted loci in single blastocysts, in this case, human blastocysts. Future developments of the protocol will allow DNA methylation analysis of a more extensive panel of genes for each embryo and at the same time, since the protocol allows for the extraction of mRNA from the embryo, the comparison between DNA methylation and gene expression
AB - Extensive epigenetic reprogramming occurs during mammalian gametogenesis and preimplantation development. DNA methylation patterns that are laid down during these stages are essential for subsequent normal foetal development. The requirement for more precise assessment of the epigenetic programming of in vitro-derived human preimplantation embryo has become of paramount importance following the identification of epigenetic diseases that are associated with assisted reproduction and/or infertility. Such techniques are also useful and applicable to experimental reproductive biology. In order to expand our knowledge of epigenetic marks, including DNA methylation, during mammalian reproduction and early development, it is necessary to test new and sufficiently sensitive protocols. There are, however, unique challenges to obtain DNA methylation data from the small cell numbers that are present in the preimplantation embryo. In this protocol, we describe the successful application of Pyrosequencing(®) to yield quantitative DNA methylation data over several CpG sites at differentially methylated regions (DMRs) at imprinted loci in single blastocysts, in this case, human blastocysts. Future developments of the protocol will allow DNA methylation analysis of a more extensive panel of genes for each embryo and at the same time, since the protocol allows for the extraction of mRNA from the embryo, the comparison between DNA methylation and gene expression
UR - http://dx.doi.org/10.1007/978-1-4939-2715-9_19
U2 - 10.1007/978-1-4939-2715-9_19
DO - 10.1007/978-1-4939-2715-9_19
M3 - Chapter or section
SN - 9781493927142
T3 - Methods in Molecular Biology
SP - 259
EP - 270
BT - Pyrosequencing
A2 - Lehmann, U.
A2 - Tost, J.
PB - Springer
CY - New York, U. S. A.
ER -
