Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber

Methods in Molecular Biology
Livia V BayerDiana P Bratu

Abstract

Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolution than ever before using light microscopy. Here we describe the steps implemented to achieve the visualization of individual messenger RNAs (mRNA) using single-molecule FISH (smFISH) probes, as well as detection of mRNA/protein (mRNP) complexes via smFISH in combination with IF.

Citations

Aug 1, 2019·Biology Open·Manja OmerzuMadelon M Maurice
Jun 17, 2020·Wiley Interdisciplinary Reviews. RNA·Fatu Badiane MarkeyMona Batish
Sep 19, 2019·Current Protocols in Neuroscience·Mona Batish, Sanjay Tyagi
Mar 23, 2019·Frontiers in Genetics·Sarah C Hughes, Andrew J Simmonds
Feb 7, 2021·Proceedings of the National Academy of Sciences of the United States of America·Aravinth Kumar JayabalanAnthony K L Leung

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