Abstract
Researchers whose experimental models are mammalian oocytes and preimplantation embryos are often limited by the yield of nucleic acids that can be isolated from such a small sample size. In addition, the limited number of cells from these types of samples makes the simultaneous recovery of RNA and DNA very difficult and often sample pooling is necessary to increase nucleic acid yield. Here we report a simple set of procedures using commercially available kits that results in consistent yield and quality of nucleic acids. After sample lysis, RNA is isolated and converted to a reusable cDNA library. Following RNA isolation, DNA is precipitated, isolated, and bisulfite converted for DNA methylation studies. Our results demonstrate the feasibility of isolating RNA and DNA from a small number of cells with repeatability of results.
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Acknowledgments
The original procedures for RNA isolation from single mouse embryos were first published by Dr. Melissa Mann (6) while in the laboratory of Dr. Marisa Bartolomei at the University of Pennsylvania. Dr. John Huntriss, University of Leeds, provided information of commercial kit for isolation of DNA from single embryos. The data used in Fig. 1 was generated by Mr. Franklin Echevarria.
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© 2012 Springer Science+Business Media, LLC
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Huffman, S.R., Almamun, M., Rivera, R.M. (2012). Isolation of RNA and DNA from Single Preimplantation Embryos and a Small Number of Mammalian Oocytes for Imprinting Studies. In: Engel, N. (eds) Genomic Imprinting. Methods in Molecular Biology, vol 925. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-011-3_13
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DOI: https://doi.org/10.1007/978-1-62703-011-3_13
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-010-6
Online ISBN: 978-1-62703-011-3
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