Advertisement

Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization

  • Brice Moukengue
  • Jérôme Amiaud
  • Camille Jacques
  • Céline Charrier
  • Benjamin Ory
  • Francois LamoureuxEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1914)

Abstract

The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE ) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes.

Key words

Bone Bone sarcoma Gene expression RT-qPCR Primers Chromogenic in situ hybridization Histology DNA mRNA miRNA LNA probes 

Notes

Acknowledgment

Thanks to www.servier.fr for the free-access’ to the clipart used.

References

  1. 1.
    Tajadini M, Panjehpour M, Javanmard SH (2014) Comparison of SYBR green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res 3Google Scholar
  2. 2.
    Cassidy A, Jones J (2014) Developments in in situ hybridisation. Methods 70(1):39–45CrossRefGoogle Scholar
  3. 3.
    Ogasawara T et al (2004) In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue. J Periodontal Res 39(1):42–49CrossRefGoogle Scholar
  4. 4.
    Shwartz Y., Zelzer E. (2014) Nonradioactive in situ hybridization on skeletal tissue sections. In: Hilton MJ (ed). Skeletal development and repair, mcxxx. Humana Press, Totowa, NJ, p. 203–215Google Scholar
  5. 5.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45CrossRefGoogle Scholar
  6. 6.
    Arabi L et al (2014) Upregulation of the miR-17-92 cluster and its two paraloga in osteosarcoma—reasons and consequences. Genes Cancer 5:56–63. https://doi.org/10.18632/genesandcancer.6 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Allen-Rhoades W et al (2015) Cross-species identification of a plasma microRNA signature for detection, therapeutic monitoring, and prognosis in osteosarcoma. Cancer Med 4:977–988. https://doi.org/10.1002/cam4.438 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chen R, Li X, He B, Hu W (2017) MicroRNA-410 regulates autophagy-related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma. Mol Med Rep. https://doi.org/10.3892/mmr.2017.6149 CrossRefGoogle Scholar
  9. 9.
    Brennan MA et al (2014) Pre-clinical studies of bone regeneration with human bone marrow stromal cells and biphasic calcium phosphate. Stem Cell Res Ther 5(5):114CrossRefGoogle Scholar
  10. 10.
    Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S, Hellemans J, Speleman F, Vandesompele J (2011) Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 39:e63CrossRefGoogle Scholar
  11. 11.
    Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P, Eveno E, Mueller O, Schroeder A, Auffray C (2005) Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res 33:e56CrossRefGoogle Scholar
  12. 12.
    D’Amico F, Skarmoutsou E, Stivala F (2009) State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods 341:1–2, 1–18CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Brice Moukengue
    • 1
  • Jérôme Amiaud
    • 1
  • Camille Jacques
    • 1
  • Céline Charrier
    • 1
  • Benjamin Ory
    • 1
  • Francois Lamoureux
    • 1
    Email author
  1. 1.INSERM, UMR1238, Bone Sarcoma and Remodeling of Calcified Tissues, Université de Nantes, Nantes Atlantique UniversitésNantesFrance

Personalised recommendations