A practical approach to FRET-based PNA fluorescence in situ hybridization.
Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed 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 as FRET efficiency measure. The 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. In this paper, we explain the FP-FISH technique workflow for reliable and reproducible results.
Blanco
Ana M
AM
Sistemas Genómicos S.L., Parque Tecnológico de Valencia, Ronda G. Marconi 6, Paterna, Spain.
Artero
Rubén
R
D017398:Alternative Splicing; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D009693:Nucleic Acid Hybridization / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results
D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; C477371:locked nucleic acid