Skip to main content
SpringerLink
Log in
Book cover

The Extracellular Matrix pp 21–31Cite as

Method for Studying ECM Expression: In Situ RT-PCR

  • Protocol

  • 2568 Accesses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1952))

Abstract

The gene expression of the extracellular matrix macromolecules is critical in the analysis of various pathologies. The use of a RT-PCR directly on a fixed tissue enables the recognition of the real expressing cells for any ECM molecules together with the tissue localization. The method here described is easy to perform using the same material as for common immunostaining and the same primers used for quantitative RT-PCR. Moreover, the used primers, designed with a final amplicon that spans the exon-exon junction, allow to detect the cDNA but not the gDNA sequences.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Rucklidge GJ, Milne G, McGaw BA, Milne E, Robins SP (1992) Turnover rates of different collagen types measured by isotope ratio mass spectrometry. Biochim Biophys Acta 1156(1):57–61

    Article  CAS  Google Scholar 

  2. Fraser JR, Laurent TC, Laurent UB (1997) Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 242(1):27–33

    Article  CAS  Google Scholar 

  3. Moriondo A, Solari E, Marcozzi C, Negrini D (2015) Diaphragmatic lymphatic vessel behavior during local skeletal muscle contraction. Am J Physiol Heart Circ Physiol 308(3):H193–H205

    Article  CAS  Google Scholar 

  4. Negrini D, Marcozzi C, Solari E, Bossi E, Cinquetti R, Reguzzoni M et al (2016) Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics. Am J Physiol Heart Circ Physiol 311(4):H892–H903

    Article  Google Scholar 

  5. Kivelä R, Salmela I, Nguyen YH, Petrova TV, Koistinen HA, Wiener Z et al (2016) The transcription factor Prox1 is essential for satellite cell differentiation and muscle fibre-type regulation. Nat Commun 7:13124

    Article  Google Scholar 

  6. Papoutsi M, Dudas J, Becker J, Tripodi M, Opitz L, Ramadori G et al (2007) Gene regulation by homeobox transcription factor Prox1 in murine hepatoblasts. Cell Tissue Res 330(2):209–220

    Article  CAS  Google Scholar 

  7. Foskett AM, Ezekiel UR, Trzeciakowski JP, Zawieja DC, Muthuchamy M (2011) Hypoxia and extracellular matrix proteins influence angiogenesis and lymphangiogenesis in mouse embryoid bodies. Front Physiol 2:103

    Article  CAS  Google Scholar 

  8. Filpa V, Bistoletti M, Caon I, Moro E, Grimaldi A, Moretto P et al (2017) Changes in hyaluronan deposition in the rat myenteric plexus after experimentally-induced colitis. Sci Rep 7(1):17644

    Article  Google Scholar 

  9. Viola M, Bartolini B, Vigetti D, Karousou E, Moretto P, Deleonibus S et al (2013) Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells. J Biol Chem 288(41):29595–29603

    Article  CAS  Google Scholar 

  10. Kramer MF, Coen DM (2001) Enzymatic amplification of DNA by PCR: standard procedures and optimization. Curr Protoc Cell Biol Appendix 3:Appendix 3F

    CAS  PubMed  Google Scholar 

  11. Kessler C (2000) Nonradioactive analysis of biomolecules, 2nd edn. Springer, Berlin, p xvii, 748

    Book  Google Scholar 

  12. Afonina I, Zivarts M, Kutyavin I, Lukhtanov E, Gamper H, Meyer RB (1997) Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res 25(13):2657–2660

    Article  CAS  Google Scholar 

  13. Bagasra O (2008) In situ polymerase chain reaction and hybridization to detect low-abundance nucleic acid targets. Curr Protoc Mol Biol Chapter 14:Unit 14.8

    PubMed  Google Scholar 

  14. Burry RW (2011) Controls for immunocytochemistry: an update. J Histochem Cytochem 59(1):6–12

    Article  CAS  Google Scholar 

  15. Cordes T, Maiser A, Steinhauer C, Schermelleh L, Tinnefeld P (2011) Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy. Phys Chem Chem Phys 13(14):6699–6709

    Article  CAS  Google Scholar 

  16. Vigetti D, Viola M, Karousou E, Deleonibus S, Karamanou K, De Luca G et al (2014) Epigenetics in extracellular matrix remodeling and hyaluronan metabolism. FEBS J 281(22):4980–4992

    Article  CAS  Google Scholar 

  17. Nuovo GJ (1995) In situ PCR: protocols and applications. PCR Methods Appl 4(4):S151–S167

    Article  CAS  Google Scholar 

  18. Dell’Orbo C, Quacci D, Raspanti M, Congiu T, Reguzzoni M, Protasoni M (2010) Atherosclerotic alterations in human carotid observed by scanning electron microscopy. Ital J Anat Embryol 115(1–2):53–58

    PubMed  Google Scholar 

  19. Zhang J, Li D, Chen D, Murchie AI (2017) Osmium tetroxide as a probe of RNA structure. RNA 23(4):483–492

    Article  CAS  Google Scholar 

  20. Lorenz TC (2012) Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp 63:e3998

    Google Scholar 

  21. Nuovo GJ (1996) The foundations of successful RT in situ PCR. Front Biosci 1:c4–c15

    Article  CAS  Google Scholar 

  22. Nuovo GJ, Gorgone GA, MacConnell P, Margiotta M, Gorevic PD (1992) In situ localization of PCR-amplified human and viral cDNAs. PCR Methods Appl 2(2):117–123

    Article  CAS  Google Scholar 

  23. Jansen RP (2001) mRNA localization: message on the move. Nat Rev Mol Cell Biol 2(4):247–256

    Article  CAS  Google Scholar 

  24. Sanford JR, Caceres JF (2004) Pre-mRNA splicing: life at the centre of the central dogma. J Cell Sci 117(Pt 26):6261–6263

    Article  CAS  Google Scholar 

Download references

Acknowledgments

E.C. is a Ph.D. student of the “Biotechnology, Biosciences and Surgical Technology” course at Università degli Studi dell’Insubria. We thank the Insubria Human Physiology Lab for the specimens used to set up the method.

Author information

Author notes

  1. Elena Caravà and Cristiana Marcozzi contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine and Surgery, University of Insubria, Varese, Italy

    Elena Caravà, Cristiana Marcozzi, Barbara Bartolini, Marcella Reguzzoni, Paola Moretto, Ilaria Caon, Evgenia Karousou, Alberto Passi & Manuela Viola

Authors
  1. Elena Caravà
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cristiana Marcozzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Barbara Bartolini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcella Reguzzoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paola Moretto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ilaria Caon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Evgenia Karousou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alberto Passi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Manuela Viola
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manuela Viola .

Editor information

Editors and Affiliations

  1. Department of Medicine and Surgery, University of Insubria, Varese, Italy

    Davide Vigetti

  2. Department of Chemistry, University of Patras, Patras, Greece

    Achilleas D. Theocharis

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Caravà, E. et al. (2019). Method for Studying ECM Expression: In Situ RT-PCR. In: Vigetti, D., Theocharis, A.D. (eds) The Extracellular Matrix. Methods in Molecular Biology, vol 1952. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9133-4_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9133-4_2

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9132-7

  • Online ISBN: 978-1-4939-9133-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation