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 .
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References
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 3
Cassidy A, Jones J (2014) Developments in in situ hybridisation. Methods 70(1):39–45
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):42–49
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. 203–215
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45
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
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
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
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):114
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:e63
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:e56
D’Amico F, Skarmoutsou E, Stivala F (2009) State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods 341:1–2, 1–18
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Moukengue, B., Amiaud, J., Jacques, C., Charrier, C., Ory, B., Lamoureux, F. (2019). Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization. In: Idris, A. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 1914. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8997-3_9
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DOI: https://doi.org/10.1007/978-1-4939-8997-3_9
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