Simultaneous RNA–DNA FISH in Mouse Preimplantation Embryos

  • Aristea Magaraki
  • Agnese Loda
  • Joost Gribnau
  • Willy M. BaarendsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1861)


Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique that allows the visualization and quantification of RNA and DNA molecules in different cellular contexts. In general, FISH applications help to advance research, cytogenetics, and diagnostics. DNA FISH can be applied, for example, for gene mapping and for detecting genetic aberrations. RNA FISH provides information about gene expression. However, in cases where RNA and DNA molecules need to be detected in the same sample, the result is often compromised by the fact that the tissue sample is damaged due to the multitude of processing steps that are required for each application. In addition, the sequential application of RNA and DNA FISH protocols on the same sample is very time consuming. Here we describe a brief protocol that enables the combined and simultaneous detection of Xist RNA and centromeric DNA of chromosome 6 in mouse preimplantation embryos. In addition, we describe how to generate indirect-labeled probes starting from BACs. This protocol may be applied to any combination of RNA and DNA detection.

Key words

RNA and DNA FISH Combination of RNA and DNA FISH Fluorescent in situ hybridization (FISH) Mouse preimplantation embryos Xist FISH X chromosome inactivation Long noncoding RNA FISH DNA FISH Long noncoding RNAs 


  1. 1.
    Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. Scholar
  2. 2.
    Kwon S (2013) Single-molecule fluorescence in situ hybridization: quantitative imaging of single RNA molecules. BMB Rep 46(2):65–72CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Hart SM, Basu C (2009) Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana. J Biomol Tech 20(2):96–100PubMedPubMedCentralGoogle Scholar
  4. 4.
    Said HM (2012) Biotin: biochemical, physiological and clinical aspects. Subcell Biochem 56:1–19. Scholar
  5. 5.
    Clemson CM, McNeil JA, Willard HF, Lawrence JB (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol 132(3):259–275CrossRefGoogle Scholar
  6. 6.
    Brockdorff N, Turner BM (2015) Dosage compensation in mammals. Cold Spring Harb Perspect Biol 7(3):a019406. Scholar
  7. 7.
    Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303(5658):644–649. Scholar
  8. 8.
    Wutz A (2011) Gene silencing in X-chromosome inactivation: advances in understanding facultative heterochromatin formation. Nat Rev Genet 12(8):542–553. Scholar
  9. 9.
    Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S, Otte AP, Brockdorff N (2004) Reactivation of the paternal X chromosome in early mouse embryos. Science 303(5658):666–669. Scholar
  10. 10.
    Marahrens Y, Panning B, Dausman J, Strauss W, Jaenisch R (1997) Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev 11(2):156–166CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Takagi N, Sugawara O, Sasaki M (1982) Regional and temporal changes in the pattern of X-chromosome replication during the early post-implantation development of the female mouse. Chromosoma 85(2):275–286CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Takizawa T, Meaburn KJ, Misteli T (2008) The meaning of gene positioning. Cell 135(1):9–13. Scholar
  13. 13.
    Chaumeil J, Augui S, Chow JC, Heard E (2008) Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation. Methods Mol Biol 463:297–308. Scholar
  14. 14.
    Namekawa SH, Lee JT (2011) Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos. Nat Protoc 6(3):270–284. Scholar
  15. 15.
    Bartlett JM (2004) Fluorescence in situ hybridization: technical overview. Methods Mol Med 97:77–87. Scholar
  16. 16.
    Sommerlad C, Mehraein Y, Giersberg M, Marben K, Rieder H, Rehder H (2002) Formalin-fixed and paraffin-embedded tissue sections. In: Rautenstrauss B, Liehr TH (eds) FISH technology (springer lab manuals). Springer, Berlin, Heidelberg, New York, pp 149–161Google Scholar
  17. 17.
    Sarvari A, Naderi MM, Sadeghi MR, Akhondi MM (2013) A technique for facile and precise transfer of mouse embryos. Avicenna J Med Biotechnol 5(1):62–65PubMedPubMedCentralGoogle Scholar
  18. 18.
    Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory press, Cold Spring HarborGoogle Scholar
  19. 19.
    Veuskens J, Hinnisdaels A, Mouras A (1993) In situ hybridization to plant metaphase chromosomes: radioactive and non-radioactive detection of repetitive and low copy number genes. In: Lindsey K (ed) Plant tissue culture manual, vol D8. Springer, New York, pp 1–15Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Aristea Magaraki
    • 1
  • Agnese Loda
    • 2
    • 3
  • Joost Gribnau
    • 1
  • Willy M. Baarends
    • 1
    Email author
  1. 1.Department of Developmental BiologyErasmus University Medical CenterRotterdamThe Netherlands
  2. 2.Mammalian Developmental Epigenetics groupInstitut CurieParis Cedex 05France
  3. 3.Department of Developmental BiologyErasmus University Medical CenterRotterdamThe Netherlands

Personalised recommendations