Chapter title |
RNA-FISH to Study Regulatory RNA at the Site of Transcription
|
---|---|
Chapter number | 12 |
Book title |
Promoter Associated RNA
|
Published in |
Methods in molecular biology, March 2017
|
DOI | 10.1007/978-1-4939-6716-2_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6714-8, 978-1-4939-6716-2
|
Authors |
Marta Soler, Raquel Boque-Sastre, Sonia Guil |
Editors |
Sara Napoli |
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. |
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