Simultaneous Detection of Nuclear and Cytoplasmic RNA Variants Utilizing Stellaris® RNA Fluorescence In Situ Hybridization in Adherent Cells

  • Sally R. Coassin
  • Arturo V. OrjaloJr.
  • Sheila J. Semaan
  • Hans E. JohanssonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool for the detection and localization of RNA and is increasingly becoming an important complement to other gene expression analysis methods. We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple RNA gene products and RNA variants in fixed mammalian cells. The technique utilizes fluorescently pre-labeled, short DNA oligonucleotides (20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in punctate fluorescent signals representing individual RNA molecules without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the quantification of single RNA transcripts. Additionally, by utilizing probe sets with spectrally distinct fluorophores, multiplex analysis of specific RNAs, or RNA variants, can be achieved. We focus on the detection of a cytoplasmic mRNA and a nuclear long noncoding RNA to illustrate the benefits of this method for cell-specific detection and subcellular localization. Post-processing of images and spot counting is briefly discussed to demonstrate the capabilities of this method for the statistical analysis of RNA molecule number per cell, which is information that can be utilized to determine overall gene expression levels and cell-to-cell gene expression variation.

Key words

Exon Intron Fluorescence In situ hybridization FISH Single molecule detection lncRNA mRNA Gene expression 



We gratefully acknowledge the continual support of Dr. Ron Cook and members of the Stellaris team at Biosearch Technologies, Inc. This chapter is dedicated to Dr. Jerry L. Ruth, a pioneer in the creation of probes for in situ hybridization.


For research use only. Not for use in diagnostic procedures. Stellaris® is a trademark of Biosearch Technologies, Inc. Products and technologies appearing in this application note may have trademark or patent restrictions associated with them. Please see for a full legal disclosure.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Sally R. Coassin
    • 1
  • Arturo V. OrjaloJr.
    • 1
  • Sheila J. Semaan
    • 1
  • Hans E. Johansson
    • 1
    Email author
  1. 1.Biosearch Technologies, Inc.2199 S. McDowell BlvdPetalumaUSA

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