Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ Hybridization.

PMID:34694613

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Cited by: 1

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Abstract

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.

Keywords

smiFISH

smFISH

Fluorescence in situ hybridization

Fluorescence microscopy

RNA imaging

RNA localization

RNA transport

Single-molecule RNA quantification

MeSH terms

Fluorescent Dyes

In Situ Hybridization, Fluorescence

Nanotechnology

RNA

RNA, Messenger

Authors

Arora, Ankita

Goering, Raeann

Velez, Pedro Tirado

Taliaferro, J Matthew

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