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The Nucleus pp 403-413 | Cite as

Visualisation of RNA by Electron Microscopic In Situ Hybridisation

  • Jacques Rouquette
  • Karl-Henning Kalland
  • Stanislav Fakan
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

Visualisation of RNA at an ultrastructural level represents a major approach to study organisation and function of the cell nucleus. In addition to methods allowing one to visualise a general distribution of RNA-containing structural constituents, in situ hybridisation (ISH) is a powerful tool for revealing specific RNA sequences or species. In this chapter we describe a method for detecting RNA by electron microscopic in situ hybridisation (EMISH) using anti-sense RNAs as probes. We first present the protocol for preparation of anti-sense RNA probes labeled with different markers, and then ossibility to evaluate the signal quantitatively. The method can also be combined with cytochemical techniques sucdescribe how such probes are applied to ultrathin sections by a method of ultrastructural ISH. The great advantage of this method is that it does not require denaturing either the specimen or the probe, thus allowing nuclear fine structure to be well preserved. The presence of the marker in the probe can be detected by immunoelectron microscopy using colloidal goldconjugated antibodies, offering the ph as EDTA staining for preferential visualisation of ribonucleoprotein-containing nuclear structural components.

Keywords

Electron microscopic in situ hybridisation RNA probes Ultrastructural analysis EDTA staining 

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Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Jacques Rouquette
    • 1
  • Karl-Henning Kalland
    • 2
  • Stanislav Fakan
    • 1
  1. 1.Centre of Electron MicroscopyUniversity of LausanneLausanneSwitzerland
  2. 2.Centre for Research in VirologyUniversity of Bergen, The Gade InstituteBergenNorway

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