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Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells

View ORCID ProfileWen Xiao, View ORCID ProfileKyu-Hyeon Yeom, View ORCID ProfileChia-Ho Lin, View ORCID ProfileDouglas L. Black
doi: https://doi.org/10.1101/2023.01.10.523484
Wen Xiao
aDepartment of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
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  • ORCID record for Wen Xiao
Kyu-Hyeon Yeom
aDepartment of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
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Chia-Ho Lin
aDepartment of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
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Douglas L. Black
aDepartment of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
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  • For correspondence: dougb@microbio.ucla.edu
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Abstract

Fluorescence In Situ Hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. Here, we present an improved method for FISH probe production that yields high purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, Amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The Degree Of Labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO and Alexa dyes) was mostly greater than 90%, comparable to commercial probes. The ease and low cost of production allowed generation of probe-sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1. Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Competing Interest Statement: The authors declare no conflict of interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 11, 2023.
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Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells
Wen Xiao, Kyu-Hyeon Yeom, Chia-Ho Lin, Douglas L. Black
bioRxiv 2023.01.10.523484; doi: https://doi.org/10.1101/2023.01.10.523484
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Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells
Wen Xiao, Kyu-Hyeon Yeom, Chia-Ho Lin, Douglas L. Black
bioRxiv 2023.01.10.523484; doi: https://doi.org/10.1101/2023.01.10.523484

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