title: RNA-FISH to Study Regulatory RNA at the Site of Transcription.
abstract: The increasing role of all types of regulatory RNAs in the orchestration of cellular programs has enhanced the development of a variety of techniques that allow its precise detection, quantification, and functional scrutiny. Recent advances in imaging and fluoresecent in situ hybridization (FISH) methods have enabled the utilization of user-friendly protocols that provide highly sensitive and accurate detection of ribonucleic acid molecules at both the single cell and subcellular levels. We herein describe the approach originally developed by Stellaris®, in which the target RNA molecule is fluoresecently labeled with multiple tiled complementary probes each carrying a fluorophore, thus improving sensitivity and reducing the chance of false positives. We have applied this method to the detection of nascent RNAs that partake of special regulatory structures called R loops. Their growing role in active gene expression regulation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012; Ginno et al., Mol Cell 45:814-825, 2012; Sun et al., Science 340:619-621, 2013; Bhatia et al., Nature 511:362-365, 2014) imposes the use of a combination of in vivo and in vitro techniques for the detailed analysis of the transcripts involved. Therefore, their study is a good example to illustrate how RNA FISH, combined with transcriptional arrest and/or cell synchronization, permits localization and temporal characterization of potentially regulatory RNA sequences.
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Soler
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Boque-Sastre
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Guil
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D038621:Regulatory Sequences, Ribonucleic Acid; D014158:Transcription, Genetic
chemical_list: D038621:Regulatory Sequences, Ribonucleic Acid; D012313:RNA
keywords: Intron; Nascent transcript; R loop; RNA-FISH; Vimentin