Nucleic Acids Research Advance Access originally published online on June 26, 2009
Nucleic Acids Research 2009 37(17):e116; doi:10.1093/nar/gkp551
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Nucleic Acids Research, 2009, Vol. 37, No. 17 e116
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization
1Sistemas Genómicos S.L., Parque Tecnológico de Valencia, Ronda G. Marconi 6, E-46980 Paterna,2Centro de Biología Molecular Severo Ochoa (CBMSO – CSIC UAM), Cantoblanco 28049 Madrid and 3Department of Genetics, University of Valencia, Dr. Moliner, 50, E-46100 Burjassot, Spain
*To whom correspondence should be addressed. Tel: +34 96 3543005; Fax: +34 96 3543029; Email: ruben.artero{at}uv.es
Received April 21, 2009. Revised June 10, 2009. Accepted June 11, 2009.
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.