ArticleNucleic acids research2009
A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.
Article in Nucleic acids research, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed, 22 citations in OpenAlex.
- Fluorinated lipid nanoparticles enable real-time tracking of mRNA delivery and uncover spatiotemporal mechanisms of immune activation.Proceedings of the National Academy of Sciences of the United States of America · 2026Article
- Explorations into Peptide Nucleic Acid Contrast Agents as Emerging Scaffolds for Breakthrough Solutions in Medical Imaging and Diagnosis.ACS omega · 2021Review
- Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications.Beilstein journal of organic chemistry · 2021Review
- Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.Nature communications · 2015Article
- Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.Nature protocols · 2013Article
- Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.Organic & biomolecular chemistry · 2013Article
- Probing nucleic acid interactions and pre-mRNA splicing by Förster Resonance Energy Transfer (FRET) microscopy.International journal of molecular sciences · 2012Review
- Visualization of the Activity of Rac1 Small GTPase in a Cell.Acta histochemica et cytochemica · 2010Article
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Authors and funding
5 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5-11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA-RNA interactions.
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