Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ Hybridization.
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Advancements in imaging technologies, especially approaches that allow the imaging of single RNA molecules, have opened new avenues to understand RNA regulation, from synthesis to decay with high spatial and temporal resolution. Here, we describe a protocol for single-molecule fluorescent in situ hybridization (smFISH) using three different approaches for synthesizing the fluorescent probes. The three approaches described are commercially available probes, single-molecule inexpensive FISH (smiFISH), and in-house enzymatically labeled probes. These approaches offer technical and economic flexibility to meet the specific needs of an experiment. In addition, we provide a protocol to perform automated smFISH spot detection using the software FISH-quant.


Fluorescence in situ hybridization
Fluorescence microscopy
RNA imaging
RNA localization
RNA transport
Single-molecule RNA quantification

MeSH terms

Fluorescent Dyes
In Situ Hybridization, Fluorescence
RNA, Messenger


Arora, Ankita
Goering, Raeann
Velez, Pedro Tirado
Taliaferro, J Matthew

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